CN101365783B - Production of glycoproteins with reduced o-glycosylation - Google Patents

Abstract

Methods of producing protein compositions having reduced amounts of O-linked glycosylation are described. The method includes producing the protein in cells cultured in the presence of an inhibitor of Pmt-mediated O-linked glycosylation and/or in the presence of one or more alpha-1, 2-mannosidase enzymes.

Description

Generation with glycosylated glycoprotein of O-of reduction

Background of invention

(1) invention field

The present invention relates to produce composition and the method for the protein with specific glycosylation pattern.Especially, the present invention relates to produce composition and the method for the glycosylated protein of the O-connection with reduction.

(2) explanation of correlation technique

Glycoprotein is the many essential functions of mediation in human and other Mammalss, comprise catalysis, signal transduction, cell-cell communication, and molecular recognition and association.In eukaryote glycoprotein constituted non-cytoplasmic protein major portion (Lis and Sharon, 1993, Eur.J.Biochem.218:1-27).At nearest 20 years, many glycoprotein were used and have been used for the treatment of purpose, and the reorganization version of the glycoprotein of natural generation becomes the major portion of biotechnological industries.The example that is used as the recombinant glycosylated protein of therapeutical agent comprises erythropoietin (EPO), therapeutic monoclonal antibodies (mAbs), tissue plasminogen activator (tPA), interferon-beta (IFN-β), granulocyte-macrophage colony stimutaing factor (GM-CSF) and human chorionic gonadotrophin (hCH) (Gumminget al., 1991, Glycobiology 1:115-130).Along with the recombinant protein that produces as potential prevention reagent and treatment reagent near clinical, the variation in the glycosylation pattern of the glycoprotein of recombinant production is themes of a large amount of concerns recently in scientific circles.

Usually, the glycosylation structure of glycoprotein oligose will depend on to produce their host species cell and change.The human cytokines that produces in the non-human host cell may contain the non-human glycosylation, and it may cause immunogenic response in the mankind, for example, and the super mannose groupization in the yeast (Ballou, 1990, Methods Enzymol.185:440-470); α in the plant (1,3)-trehalose and β (1,2)-wood sugar (Cabanes-Macheteau et al., 1999, Glycobiology, 9:365-372); N-n acetylneuraminic acid n in the Chinese hamster ovary cell (Noguchi et al., 1995.J.Biochem.117:5-62); And, the Gal α-1 in the mouse, and the 3Gal glycosylation (Borrebaeck etal., 1993, Immun.Today, 14:477-479).In zooblast, comprise that with the carbohydrate chain of protein bound the N-glucosides of bonding carbohydrate chain that l-asparagine in protein (Asn) residue is combined (is also referred to as the N-glycan; Or the glycosylation that connects of N-) the O-glycosides of bonding carbohydrate chain that Serine (Ser) or Threonine (Thr) residue are combined and in protein (is also referred to as the O-glycan; Or the glycosylation of O-connection).

Because the oligose structure of the glycoprotein that non-human mammal produces is tended to relevant more closely with those of human glycoprotein, so the glycoprotein of most of commerce is produced in mammalian cell.Yet mammalian cell has several important shortcomings as the host cell of protein production.Except the expense height, the process of marking protein has produced the heterogeneous population of sugar form in mammalian cell, has low volume titre, and needs ongoing virus prevention and plenty of time to produce stable clone.

Should be understood that, specific sugar form (glycoform) on the protein may have a deep effect on the character of protein, pharmacokinetics, drug effect, acceptor interaction and the tissue specificity target character (Graddis et al., 2002.Curr Pharm Biotechnol.3:285-297) that comprise it.For example, be associated (Jefferis and Lund, 1997, Antibody Eng.Chem.Immunol., 65:111-128 of the different glycosylation pattern that has shown Ig and different biological property; Wright and Morrison, 1997, Trends Biotechnol., 15:26-32).What further show is, the galactosylation of glycoprotein may change with cell culture condition, it depends on that the specific semi-lactosi pattern on the glycoprotein may make that some glycoprotein compositions is immunogenic (Patel etal., 1992.Biochem is J.285:839-845).Yet, owing to know which specific sugar form the biological function of expectation not to be contributed, the ability of the specific sugar form on the enrichment glycoprotein is high expectations.Because different sugar forms is relevant with different biological properties, the ability that enrichment has the glycoprotein of specific sugar form can be used for illustrating the specific sugar form of glycoprotein and the relation between the special biological.And the ability that enrichment has the glycoprotein of specific sugar form allows to produce the therapeutic glycoprotein with particular characteristics.Thereby the glycoprotein compositions that produces for specific sugar form enrichment is high expectations.

Though the glycosylated approach that N-connects has become the target of macromethod, the glycosylated process that O-connects and function are not also understood well.Yet, be known that the glycosylation that is connected with N-compares, the O-glycosylation is translation back event, its in the cis-golgi body, take place (Varki, 1993, Glycobiol., 3:97-130).Though the glycosylated total receptor sequence that the glycosylation that connects as N-connects for O-appears to and does not exist, the frequency of the raising that has relatively shown position-1 with respect to glycosylated residues and+3 place's proline residues of aminoacid sequence and Serine, Threonine and alanine residue significantly improves (Wilson et al. around the glycosylation site that many O-of several glycoprotein connect, 1991, Biochem.J., 275:529-534).The extension of Serine and threonine residues also may be the glycosylated potential site of O-in glycoprotein.

A gene family that plays effect in the glycosylation that O-connects is encoding D ol-P-Man: the gene of albumen (Ser/Thr) mannose transferase (Pmt).The gene of these high conservatives for example for example identify in fungi etc. with lower eukaryotes at higher eucaryote by the mankind, rodent, insect etc.Yeast for example Saccharomyces cerevisiae and Pichia pastoris coding nearly the PMT gene of seven kinds of coding Pmt homologues (at Wilier et al.Curr.Opin.Struct.Biol.2003Oct; 13 (5): summarize among the 621-30).In yeast, the glycosylation that O-connects start from by one of seven kinds of O-mannose transferase genes in endoplasmic reticulum from the dolichol phosphate seminose Serine to Noviose albumen or threonine residues add initial seminose.As if though the PMT gene of seven kinds of coding Pmt homologues is arranged in yeast, and the O-mannose groupization of the mycoprotein of secreting in yeast and heterologous protein depends primarily on the gene of coding Pmt1 and Pmt2, Pmt1 and Pmt2 seem to work as heterodimer.It is high conservative that PMT1 and PMT2 and they divide other protein product Pmt1 and Pmt2 to seem between species.

People such as Tanner are in U.S. Patent No. 5,714, PMT1 and the PMT2 gene of Saccharomycescerevisiae (yeast saccharomyces cerevisiae) have been described in 377, and the use fungal cell produces the method for the glycosylated recombinant protein of the O-connection with reduction, one or more PMT genes are modified hereditarily in described fungal cell, thereby produce the glycosylated recombinant protein of the O-connection with reduction.

People such as Ng disclose in the disclosed patent application NO.20020068325 of the U.S. by using antisense or common inhibition or the through engineering approaches by the yeast host bacterial strain to suppress the O-glycosylation, and described yeast host bacterial strain has the relevant gene of the glycosylation that connects with O-, particularly the afunction mutant of one or more PMT gene aspect.

UDP-N-ethanoyl-α-D-galactosamine: polypeptide N-ethanoyl galactosyl transaminase (GalNAc-transferring enzyme) relates to the glycosylation of the Saliva Orthana type O-connection of finding in the higher eucaryote.These enzymes have started specific Serine and the amino acid whose O-glycosylation of Threonine in the protein, and by adding N-acetylgalactosamine to these amino acid whose hydroxyls, thereby mannose residue can be added in the mode of substep on described hydroxyl.People's U.S. Patent No.s 5 such as Clausen; 871,990 and the disclosed patent application NO.20050026266 of the U.S. in coding UDP-N-ethanoyl-α-D-galactosamine disclosed: the nucleic acid family of polypeptide N-ethanoyl galactosyl transaminase (GalNAc-transferring enzyme).Clausen discloses in the disclosed patent application NO.20030186850 of the U.S. and has used GalNAc-β-phenmethyl optionally to suppress the lectin of polypeptide GalNAc-transferring enzyme, and the substrate that does not serve as other glycosyltransferases that relate in the biosynthesizing of O-glycan, thereby suppress the O-glycosylation.

The glycosylated inhibition that O-connects has been described.For example, people such as Orchard are in U.S. Patent No. 7,105, have described Ben Yajiaji thiazolidinediones (thiazolidinediones) and they in 554 as antimycotic agent, for example the purposes of anti-mycotic agent.These Ben Yajiaji thiazolidinediones are reported to suppress the Pmt1 enzyme, the integrity of the formation of the sweet dew protein that prevention O-connects and infringement fungal cell wall.End-result is cellular swelling and finally dead by breaking.

People such as Konrad disclose in the disclosed patent application NO.20020128235 of the U.S. and have suppressed by pharmacology ground that O-connects in tissue or the cell Protein Glycosylation Overview is treated or the method for prevent diabetes.This method relies on to be used the N-acetylglucosamine transferase that connects in conjunction with O-and thereby suppresses glycosylated (Z)-1-[N-(3-aminopropyl)-N-(n-propyl group) amino that O-connects] diazen-ium-1,2diolate or derivatives thereof treatment diabetic individual.

People such as Kojima are in U.S. Patent No. 5,268, disclose in 364 utilize compound for example phenyl-α-N-acetylgalactosamine be used for to suppress the glycosylated therapeutic composition of O-, described compound suppresses the glycosylated extension of O-, cause the accumulation of O-α-GalNAc, come SLex or the SLea of blocking leukocyte or tumour cell to express, thereby suppress these cells to endotheliocyte and hematoblastic adhesion.

People such as Boime are in U.S. Patent No. 6,103, disclose the variant of hormone in 501, the wherein glycosylation that connects of the O-that changes by the aminoacid sequence of modifying the glycosylation site place.

The inventor finds, for fungi usually be fatal, as the specific compound of the inhibition of Pmt protein, can be using for the nonlethal mode of host cell, the glycosylated recombinant protein that connects for generation of the O-with reduction.This glycosylation that makes the O-of the protein that produced by fungi and yeast cell be connected can be controlled.The contriver thinks the compound of other classifications of the non-fatal inhibition of PMT enzyme, and the glycoprotein that has the glycosylated improvement that the O-of reduction connects for generation also is useful.The inventor further found, individually or with the chemical inhibitor of Pmt protein in combination, add the enzyme of some kind to host cell or cell culture, that is, α-1,2-mannosidase have caused the glycosylated further reduction of O-.

Summary of the invention

The invention provides the method for producing protein and glycoprotein with specific glycosylation pattern.Especially, the invention provides and in host cell, produce the recombinant protein method for compositions, wherein glycosylated one or more inhibitions that connect by the Pmt mediation/O-with protein in the described host cell contact described host cell, or with one or more α-1, the 2-mannosidase contacts described host cell or described recombinant protein, or by the two, the glycosylation that the O-of recombinant protein connects is lowered.Compare with the glycosylated quantity that the O-of the recombinant protein that produces in the described host cell under the situation that does not have described inhibition or glycoprotein is connected, the glycosylated quantity that the O-of recombinant protein or glycoprotein connects is lowered.

The glycosylation that the O-of Pmt-mediation connects refers to the glycosylation that a kind of O-connects, wherein mannose residue to the transfer of the Serine of protein or threonine residues by PMT gene or its homologue encoded protein-O-D-mannose transferase (Pmt) or homologue mediation.The glycosylated inhibition that the O-of Pmt-mediation connects comprises the inhibition of the homologue that suppresses any PMT gene.Current preferred aspect in, described inhibition suppresses Pmt1 and/or the Pmt2 activity of fungi and yeast at least, or at other organisms, includes but not limited to the activity of corresponding homologue in Mammals, plant and the insect.

Current, preferably, the glycosylated quantity that O-connects is by using the Chemical Inhibition thing, for example is called the Chemical Inhibition thing that the chemical classes of Ben Yajiaji thiazolidinedione contains and reduces.In specific embodiment, described Chemical Inhibition thing is selected from by 5-[[3,4-two (phenyl methoxyl group) phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate; 5-[[3-(1-phenyl ethoxy)-4-(2-phenyl ethoxy)] phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate; 3-hydroxyl-4-(2-phenyl ethoxy) phenyl aldehyde; 3-(1-phenyl ethoxy)-4-(2-phenyl ethoxy)-phenyl aldehyde; 5-[[3-(1-phenyl-2-hydroxyl) oxyethyl group)-and 4-(2-phenyl ethoxy)] phenyl] methylene radical]-group that 4-oxo-2-sulfo--3-thiazolidine acetate constitutes.

Further, provide the glycosylated recombinant protein method for compositions that produces the O-connection with reduction, one or more inhibitions and/or one or more α-1 of the Pmt protein that relates in its glycosylation of using O-to connect, 2-mannosidase produce the glycosylated protein of the O-connection with reduction.Current preferred α-1, the 2-mannosidase can separate from eukaryotic cell, comprises Mammals and yeast cell.In presently preferred embodiments, α-1,2-mannosidase are those that are produced by Trichoderma reesei, Saccharomyces sp. or Aspergillus sp..In other presently preferred embodiments, α-1, the 2-mannosidase can produce from the chimeric construct body, described chimeric construct body comprises coding for alpha-1, the nucleotide sequence of the catalyst structure domain of 2-mannosidase, described nucleotide sequence is operably connected with the nucleotide sequence of the cell-targeting signal peptide that coding does not link to each other with described catalyst structure domain usually.In other embodiments, α-1, the 2-mannosidase can produce and add in the cell culture individually, maybe can by with recombinant glycoprotein coexpression α-1, the 2-mannosidase produces.

Aspect this method specific, described recombinant protein composition comprises the glycosylated glycoprotein with N-connection, and wherein said recombinant glycoprotein comprises at least one main N-sugar form (predominantN-glycoform) and has the glycosylation of the O-connection of reduction.Therefore, further provide glycoprotein compositions, it comprises the N-glycan structures of main kind, and with the glycosylated inhibition or the α-1 that are not connected at the O-of Pmt-mediation, the composition of the glycoprotein that produces in the host cell of hatching under the situation of 2-mannosidase is compared, glycosylation with O-connection of reduction, described α-1,2-mannoside endonuclease capable surpasses a mannose residue from glycan structures finishing (trimming).Aspect specific, glycoprotein compositions comprises to have and is selected from by Man ₅GlcNAc ₂, Man ₃GlcNAc ₂, GlcNAcMan ₅GlcNAc ₂, GlcNAcMan ₃GlcNAc ₂, GlcNAc ₂Man ₃GlcNAc ₂, GalGlcNAcMan ₅GlcNAc ₂, Gal (GlcNAc) ₂Man ₅GlcNAc ₂, (GalGlcNAc) ₂Man ₅GlcNAc ₂, NANAGalGlcNAcMan ₃GlcNAc ₂, NANA ₂Gal ₂GlcNAcMan ₃GlcNAc ₂And GalGlcNAcMan ₃GlcNAc ₂The glycoprotein of the main N-glycan structures of the group that sugar form constitutes.An important aspect of described method is, it provides the glycosylation of the O-connection that comprises reduction and has mainly comprised the glycoprotein compositions of the sugar form of specific N-connection, wherein with respect to from the mammalian cell cultures composition of the identical glycoprotein that produces of Chinese hamster ovary celI for example, this recombinant glycoprotein can represent the biologic activity of raising and/or the unexpected immunogenicity of reduction.Generation comprise glycosylation that the O-of reduction connects and mainly other benefits of the glycoprotein compositions of the sugar form that is connected of N-be, it has avoided producing sugar form unexpected or non-activity and heterogeneous mixture, and it may be induced unexpected effect and/or dilute more effective sugar form.Thereby, mainly comprise, for example, Man ₅GlcNAc ₂, Man ₃GlcNAc ₂, GlcNAcMan ₅GlcNAc ₂, GlcNAcMan ₃GlcNAc ₂, GlcNAc ₂Man ₃GlcNAc ₂, GalGlcNAcMan ₅GlcNAc ₂, Gal (GlcNAc) ₂Man ₅GlcNAc ₂, (GalGlcNAc) ₂Man ₅GlcNAc ₂, NANAGalGlcNAcMan ₃GlcNAc ₂, NANA ₂Gal ₂GlcNAcMan ₃GlcNAc ₂And GalGlcNAcMan ₃GlcNAc ₂Sugar form, and have the curative drug composition of the glycosylated glycoprotein molecule that the O-of reduction connects also may be effective at lower dosage, thereby have higher effectiveness/tire.

Thereby, provide the glycosylated method of protein of producing the O-connection with reduction, comprise the nucleic acid that coded protein is provided; Described nucleic acid is imported the culture that host cell is provided in the host cell; Glycosylated one or more inhibitions that the O-that mediates with Pmt connects contact described culture; And the glycoprotein that is separated in host cell generation under the situation that has described inhibition produces the glycosylated protein of the O-connection with reduction.

Aspect described method specific, described culture growth time enough is to provide a large amount of host cells with described nucleic acid (a multiplicity of the host cells), glycosylated one or more inhibitions that connect with the O-of Pmt mediation contact described culture afterwards, perhaps have the described culture of growth under the situation of glycosylated one or more inhibitions that the O-of Pmt mediation connects in being established at culture.

Aspect described method further, the nucleic acid of coded protein is operably connected with inducible promoter.Then, described culture growth time enough is to provide a large amount of host cells with described nucleic acid, glycosylated one or more inhibitions that connect with the O-of Pmt mediation and the inductor of described promotor contact described substratum to induce described protein expression afterwards, and be separated in protein that host cell under the situation that has described one or more inhibitions and inductor produces and have the glycosylated protein that the O-of reduction is connected with generation, perhaps described culture contacts to induce described protein expression certain hour with the inductor of described promotor, glycosylated one or more inhibitions that connect with O-contact described culture afterwards, and are separated in described host cell produces under the situation that has described inhibition and inductor protein and have the glycosylated protein that the O-of reduction is connected with generation.

Further provide the glycosylated method of protein of producing the O-connection with reduction, comprise the nucleic acid that coded protein is provided; Described nucleic acid is imported the culture that host cell is provided in the host cell; With one or more (one or more) α-1, the 2-mannosidase contacts described culture; And be separated in and have described one or more α-1, the protein that host cell produces under the situation of 2-mannosidase produces the glycosylated glycoprotein that O-with reduction connects.

Aspect described method specific, described culture growth time enough is used one or more α-1 afterwards so that a large amount of host cells with described nucleic acid to be provided, and the 2-mannosidase contacts described culture.In other respects, described culture is grown under the situation of 2-mannosidase there being one or more α-1.

Aspect described method further, coding one or more α-1 are provided, second nucleic acid of 2-mannosidase also imports described second nucleic acid in the described host cell.Aspect specific, the coding that is operably connected to inducible promoters one or more α-1 are provided, second nucleic acid of 2-mannosidase, described second nucleic acid is imported in the described host cell, described culture growth time enough is to provide a large amount of host cells, induce described protein and described one or more α-1 afterwards, the expression of 2-mannosidase produces the glycosylated protein of the O-connection with reduction, perhaps described protein expression is induced certain hour, induce described one or more α-1 afterwards, the expression of 2-mannosidase produces the glycosylated protein of the O-connection with reduction, perhaps described one or more α-1, the expression of 2-mannosidase is induced certain hour, the glycosylated protein that the O-that induces described protein expression to produce afterwards to have reduction connects.

Further provide the glycosylated method of protein of producing the O-connection with reduction, comprise the nucleic acid that the coded protein that is operatively connected with inducible promoters is provided; The host cell that contains described nucleic acid with also growing in the described nucleic acid importing host cell produces the culture of host cell; Glycosylated one or more inhibitions and one or more α-1 that the O-that mediates with Pmt connects, the 2-mannosidase contacts described culture; And be separated in and have described one or more inhibitions and described one or more α-1, the glycoprotein that described host cell produces under the situation of 2-mannosidase produces the glycosylated protein that O-with reduction connects.

Aspect described method specific, described culture growth time enough is to provide a large amount of host cells with described nucleic acid, glycosylated one or more inhibitions that connect with O-contact described culture afterwards, perhaps have the described culture of growth under the situation of glycosylated one or more inhibitions that the O-of Pmt mediation connects in being established at culture.

Aspect described method further, the nucleic acid of coded protein is operably connected with inducible promoter.Then, described culture growth time enough is to provide a large amount of host cells with described nucleic acid, glycosylated one or more inhibitions that connect with the O-of Pmt mediation and the inductor of described promotor contact described substratum to induce described protein expression afterwards, and be separated in protein that host cell under the situation that has described one or more inhibitions and inductor produces and have the glycosylated protein that the O-of reduction is connected with generation, perhaps described culture contacts to induce described protein expression certain hour with the inductor of described promotor, glycosylated one or more inhibitions that connect with O-contact described culture afterwards, and are separated in described host cell produces under the situation that has described inhibition and inductor protein and have the glycosylated protein that the O-of reduction is connected with generation.

Aspect described method specific, described culture growth time enough is used one or more α-1 afterwards so that a large amount of host cells with described nucleic acid to be provided, and the 2-mannosidase contacts described culture.In other respects, described culture is grown under the situation of 2-mannosidase there being one or more α-1.

Aspect described method further, coding one or more α-1 are provided, second nucleic acid of 2-mannosidase also imports described second nucleic acid in the described host cell.Aspect specific, the coding that is operably connected to inducible promoters one or more α-1 are provided, second nucleic acid of 2-mannosidase, described second nucleic acid is imported in the described host cell, described culture growth time enough is to provide a large amount of host cells, induce described protein and described one or more α-1 afterwards, the expression of 2-mannosidase produces the glycosylated protein of the O-connection with reduction, perhaps described protein expression is induced certain hour, induce described one or more α-1 afterwards, the expression of 2-mannosidase produces the glycosylated protein of the O-connection with reduction, perhaps described one or more α-1, the expression of 2-mannosidase is induced certain hour, the glycosylated protein that the O-that induces described protein expression to produce afterwards to have reduction connects.

Aspect the aforesaid method of one or more inhibitions that use Pmt albumen further, current, preferably, described one or more inhibitions are selected from the molecular species that comprises the Ben Yajiaji thiazolidinedione.Current, preferably, described one or more inhibitions are selected from by 5-[[3,4-two (phenyl methoxyl group) phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate; 5-[[3-(1-phenyl ethoxy)-4-(2-phenyl ethoxy)] phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate; And 5-[[3-(1-phenyl-2-hydroxyl) oxyethyl group)-and 4-(2-phenyl ethoxy)] phenyl] methylene radical]-group that 4-oxo-2-sulfo--3-thiazolidine acetate constitutes.

Using α-1, the specific aspect of the aforesaid method of 2-mannosidase, preferably current, described α-1,2-mannosidase are selected from the group that is made of Trichoderma reesei, Saccharomyces sp. and Aspergillus sp..Current, preferably, α-1, the 2-mannosidase is from Trichoderma reesei.Alternatively, except first nucleic acid of coded protein or glycoprotein, host cell can comprise second nucleic acid that is operatively connected with inducible promoters, its coding for alpha-1,2-mannosidase.α-1, the expression of 2-mannosidase and protein or glycoprotein can side by side be induced, or at α-1, the expression of induced protein or glycoprotein before the expression of 2-mannosidase, otherwise perhaps.

Though described method can be used and produce any host cell with glycosylated protein that O-connects and carry out, current preferred aspect in, described host cell is low eukaryotic cell, preferably fungal cell or the yeast cell of waiting.Current, preferably, described host cell is selected from by the group from the cellularity of K.lactis, Pichia pastoris, Pichia methanolica and Hansenula.Particularly, in the further embodiment that produces recombinant glycoprotein, described host cell is yeast or filamentous fungal cells, and it is produced the glycoprotein that mainly has specific N-glycan structures by genetic modification.Aspect particularly preferred, described host cell is modified hereditarily, thus their express recombinant glycoprotein, and wherein glycosylation pattern is picture human (human-like) or humanized.Especially, described host cell can be modified, thereby they express the recombinant glycoprotein that mainly has specific expectation N-glycan structures.This when with glycosylation profile (glycosylation profiles) when using together with ground, the low eukaryotic host cell that waits refers to any eukaryotic cell, it produces the glycan of the N-connection that contains a large amount seminose usually, and thereby comprise most typical eukaryotic cell such as low, comprise unicellular and cellulous fungi and alga cells.

All publications, patent, patent application and other reference intactly merge them by reference referred in this.

Brief description of drawings

Accompanying drawing 1 has illustrated that the Pmt inhibition is to the glycosylated influence of O-of the reorganization reporter protein matter of secretion in Pichia pastoris.The Chemical Inhibition thing of Pmt with the O-glycosylation be reduced to the bacterial strain that lacks PMT1 in the similar level of observing.Utilize the Western trace of anti-polyhistidine antibody to be used to detect the human Kringle 1-3 structural domain (K1-3) of the His label of human plasminogen in the growth medium of wild-type (swimming lane 1-3) and pmt1 (swimming lane 4-5) bacterial strain.Slower migration band (in swimming lane 1 for K1-3 as the disperse finding of high molecular more) shows the glycosylated protein of O-.Pmti-1, PMT inhibition 1.

Accompanying drawing 2 has shown the Western trace, and it has represented the glycosylated influence of O-of T. reesei alpha-Mannosidase and the light chain immunoglobulin of Chemical Inhibition thing Pmti-2 and heavy chain polypeptide.T.reesei alpha-Mannosidase and chemical substance Pmt inhibition have all reduced the glycosylated level of O-.

Detailed description of the invention

The invention provides the method for in specific host cell, expressing the recombinant protein (comprising polypeptide and glycoprotein) of the glycosylation sensitivity that connects for O-, in this cell type, have the glycosylation quantity (comprising the glycosylation that does not have O-to connect) of the O-connection of reduction.Method of the present invention relates in protein expression is induced, there are one or more Dol-P-Man: protein (Ser/Thr) mannose transferase (Pmt) albumen or one or more α-1, the 2-mannosidase, or under the two the situation, in host cell, induce the expression of protein of interest matter, be responsive at protein described in the described host cell for the glycosylation that O-connects, described Dol-P-Man: protein (Ser/Thr) mannose transferase (Pmt) albumen relates in the cell seminose to the Serine of protein or the transfer of threonine residues.If with lacking described inhibition or one or more α-1, the glycosylation quantity that on the protein that produces under 2 mannosidases or the two the situation O-that exists is connected is compared, have described inhibition or one or more α-1, expressed protein has the glycosylation quantity of the O-connection of reduction under the situation of 2-mannosidase.Described method is useful especially, because it provides at host cell lower eukaryotes for example, for example produce the means of the relevant protein for the treatment of in yeast and the bacterium, what wherein expect is the O-glycosylation quantity that described protein has reduction, and described host cell produces the glycan that has O-and connect, the protein with glycan of the O-connection that reduces quantity usually.Yet though described method is particularly suitable for expressing the glycosylated protein of the O-connection with reduction in lower eukaryotes, described method also can be put into practice in higher eucaryote and bacterium.

Produce the glycosylated protein of the O-connection with reduction in the host cell for the glycosylation sensitivity that O-is connected at protein, described method has been compared improvement with prior art.For example, Tanner etc. are in U.S. Patent No. 5,714, described in 377 and utilized for example glycosylated method of protein of the O-connection with reduction of yeast cell generation reorganization of fungal cell, one or more PMT genes of coding Pmt albumen are modified hereditarily in described cell, thereby produce the glycosylated recombinant protein of the O-connection with reduction.Though one of deletion PMT1 or PMT2 gene allow to produce the glycosylated protein of the O-connection with reduction of recombinating in fungal host cells in fungal host cells, growth is important for host cell for PMT1 and PMT2 expression of gene, any one independent deletion all influences the ability of fungal host cells growth unfriendly, thereby the recombinant protein of the glycosylation quantity that connects of the O-that makes him be difficult to produce the host cell of sufficient amount or have reduction.It is fatal that the deletion of two genes seems for fungal host cells.Therefore, in host cell the genetics of PMT1 and PMT2 gene as if eliminate will be the unexpected means that produce the glycosylated protein that the O-with reduction of reorganization is connected.

By contrast, the PMT gene is not modified or is deleted in the host cell of Shi Yonging in the method for the invention, its allow host cell O-glycosylation those for cell growth important protein matter, in the activity inhibited of Pmt albumen.Usually, if allowing host cell to grow into the level that obtains with deletion PMT gene, this compares higher level.In addition, in specific embodiment, the expression of recombinant protein in host cell controlled by inducible promoter, Pmt activity in the host cell is not suppressed, or adds one or more α 1,2-mannosidase, or the two, be induced up to the expression of recombinant protein.This allows in the expression of inducing recombinant protein and adds Pmt inhibition and/or one or more α 1, produces a large amount of host cells of the nucleic acid that contains the recombinant protein of encoding before the 2-mannosidase in culture.With delete one or more PMT genes and bad host cell grown takes place in cultivation compares, the glycosylated recombinant protein that this O-with reduction that allows to produce bigger quantity in the shorter time in culture connects.

Relatively this improvement of prior art also is convenient to modified to produce the glycoprotein that mainly has the glycan structures that specific N-connects hereditarily but also produce the glycosylated glycoprotein that O-with reduction connects in the host cell of this glycoprotein of O-glycosylation.Produce the method for the various glycoprotein mainly have the sugar form that specific N-connects and in U.S. Patent No. 7,029,872 and the U.S. disclosed application NO.20050170452,20050260729,20040230042,20050208617,20050208617,20040171826,20060160179,20060040353 and 20060211085 in open.Any host cell of describing in aforementioned patent and patent application may be used to utilize method disclosed herein to produce and mainly has the glycosylated glycoprotein of the glycan structures that specific N-is connected and the O-connection with reduction.Do not have been found that with there being adorned host cell and compare that some is modified to produce the host cell of the glycoprotein of the glycan structures that mainly has specific N-connection hereditarily and grows so not well under given conditions in culture.For example, compare with the fungi that does not have genetic modification or yeast cell, the gene that particularly wherein relates to super mannose groupization is deleted, having added the fungi and the yeast cell growth that produce other required genes of glycan structures that specific Mammals or human sample N-connect must be so not good.In the fungi or yeast cell of these genetic modifications, the deletion of further introducing PMT1 or PMT2 gene all is fatal for cell, perhaps influences cell grows into sufficient amount in culture ability unfriendly.By inducing recombinant glycoprotein to express and add inhibition or one or more α 1 of Pmt protein-active, 2-mannosidase or the two permissive cell before grow into enough quantity in culture, produce the glycosylated recombinant glycoprotein that the O-that mainly has glycan structures that specific N-connects and reduction is connected, method herein has avoided deleting the potential harmful effect of PMT1 and PMT2 gene.

Thereby, an important aspect of described method is, it provides the glycosylation of the O-connection that comprises reduction and has mainly comprised the glycoprotein compositions of the sugar form of specific N-connection, wherein with respect to from the mammalian cell cultures composition of the identical glycoprotein that produces of Chinese hamster ovary celI for example, described recombinant glycoprotein can represent the biologic activity of raising and/or the unexpected immunogenicity of reduction.Generation comprise glycosylation that the O-of reduction connects and mainly other benefits of the glycoprotein compositions of the sugar form that is connected of N-be, it has avoided producing sugar form unexpected or non-activity and heterogeneous mixture, and this may induce unexpected effect and/or dilute more effective sugar form.Thereby, mainly comprise, for example, Man ₅GlcNAc ₂, Man ₃GlcNAc ₂, GlcNAcMan ₅GlcNAc ₂, GlcNAcMan ₃GlcNAc ₂, GlcNAc ₂Man ₃GlcNAc ₂, GalGlcNAcMan ₅GlcNAc ₂, Gal (GlcNAc) ₂Man ₅GlcNAc ₂, (GalGlcNAc) ₂Man ₅GlcNAc ₂, NANAGalGlcNAcMan ₃GlcNAc ₂, NANA ₂Gal ₂GlcNAcMan ₃GlcNAc ₂And GalGlcNAcMan ₃GlcNAc ₂Sugar form, and have the curative drug composition of the glycosylated glycoprotein molecule that the O-of reduction connects also may be effective at lower dosage, thereby have higher effectiveness/tire.

Usually, the glycosylated method of protein of producing the O-connection with reduction comprises the nucleic acid transformed host cell of using coding recombinant protein or heterologous protein, and wherein expectation is the glycosylated protein that produces the O-connection with reduction.The nucleic acid of described recombinant protein of encoding is operably connected with the adjusting sequence of the expression of allowing recombinant protein.Such adjusting sequence comprises inducible promoter, randomly comprise the upstream or 5 of the nucleic acid of encoding fusion protein ' enhanser, and 3 ' or the Transcription Termination site in downstream of the nucleic acid of coding recombinant protein.5 ' UTR zone and 3 ' non-translational region that nucleic acid is usually also encoded and had ribosome bind site.Described nucleic acid is the part of carrier normally, and described carrier is reproducible in the cell of express recombinant protein matter.Carrier also can contain marker to allow the identification cell transformed.Yet some cell type, particularly yeast can successfully transform with the nucleic acid that lacks external carrier sequence.

The nucleic acid of the recombinant protein of coding expectation can obtain from several sources.Can use at the primer of conservative region from the clone amplification cDNA sequence of known this protein of expression (referring to, Marks et al. for example, J.Mol.Biol.581-596 (1991)).Nucleic acid also can be according to the sequence de novo synthesis in the scientific literature.Nucleic acid also can be by crossing over the expectation sequence the extension of overlapping oligonucleotide synthesize (referring to, Caldas et al. for example, Protein Engineering, 13,353-360 (2000)).

In one aspect, the nucleic acid of coded protein is operably connected with inducible promoters, and described promotor allows that protein expression is induced when expectation.In yet another aspect, the nucleic acid of coded protein is operably connected with constitutive promoter.For the ease of the separation of expressed protein, preferably current, protein comprises signal sequence, and described signal sequence instructs protein secreting in cell culture medium, thereby protein can be separated therein.Aspect first, add glycosylated one or more inhibitions that O-connects to substratum before, transformed host cells is cultivated a large amount of host cells that time enough produces the expectation of the protein that is enough to produce desired amt.Inductor and inhibition can side by side add in the culture, perhaps add inductor to culture before adding one or more Pmt inhibitions, perhaps add one or more Pmt inhibitions to culture before adding inductor.The glycosylated protein of inducing that generation has the O-connection of reduction can reclaim from substratum, perhaps for the protein that does not have signal sequence, reclaims from host cell by cracking.Aspect second, wherein the nucleic acid of coded protein is operably connected with constitutive promoter, when being established, culture in substratum, adds glycosylated one or more inhibitions that O-connects, the glycosylated protein that the O-with reduction that produces connects can reclaim from substratum, or for the protein that does not have signal sequence, reclaim from host cell by cracking.The example of the method for utilizing inducible promoters is described shown in the embodiment 2, illustrates that the example of the method for utilizing constitutive promoter is shown in the embodiment 3.

The useful inhibition of glycosylated protein that has the O-connection of reduction for generation is chemical substance or the composition that suppresses one or more Pmt activity of proteins.When host cell is lower eukaryotes for example when fungi or yeast, expectation be that described inhibition suppresses Pmt1 or Pmt2 or the two activity at least.In higher eucaryote, expectation be that described inhibition suppresses in this higher eucaryote the activity corresponding to the homologue of Pmt1 or Pmt2.Operable Chemical Inhibition thing is included in U.S. Patent No. 7,105, the thiazolidinediones of identifying in 554, it comprises 5-[[3,4-two (phenyl methoxyl group) phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate; 5-[[3-(1-phenyl ethoxy)-4-(2-phenyl ethoxy)] phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate; 3-hydroxyl-4-(2-phenyl ethoxy) phenyl aldehyde; 3-(1-phenyl ethoxy)-4-(2-phenyl ethoxy)-phenyl aldehyde; And, 5-[[3-(1-phenyl-2-hydroxyl) oxyethyl group)-4-(2-phenyl ethoxy)] phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate.Other compounds that come in handy are similar compounds on the disclosed structure in WO 94/29287 such as Voss, it discloses the method for generation arylidene-4-oxo-2-sulfo--3-thiazolidine carboxylic-acid, disclosing them is useful in the prevention of the Late Effect of diabetes and treatment and atherosclerosis (atherosclerosis and arteriosclerosis) and arteriosclerotic prevention and treatment, and Esswein etc. is in U.S. Patent No. 6,673, similar compound on the disclosed structure in 816, it discloses and has produced if the method for the derivative of tannin carboxylic acid (rhodaninecarboxylic acids) and they are used for the purposes of the treatment of metabolic bone imbalance.

In an embodiment, be selected from by 5-[[3 4-two (phenyl methoxyl group) phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate; 5-[[3-(1-phenyl ethoxy)-4-(2-phenyl ethoxy)] phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate; With 5-[[3-(1-phenyl-2-hydroxyl) oxyethyl group)-4-(2-phenyl ethoxy)] phenyl] methylene radical]-the Chemical Inhibition thing of the group that 4-oxo-2-sulfo--3-thiazolidine acetate constitutes is displayed on that to produce the glycosylated recombinant protein aspect that O-with reduction is connected in the Pichiapastoris bacterial strain with complete, functional PMT1 and PMT2 gene be effective.The table 1 of embodiment 2 has shown in the culture that adds reorganization Pichia pastoris when any expression at recombinant protein of above-mentioned three kinds of Pmt Chemical Inhibition things is induced to, described reorganization Pichia pastoris has complete functional PMT1 and the PMT2 gene also can be secreted Kringle 1-3 protein with the coding reorganization, the nucleic acid that is operatively connected with inducible promoters transforms, compare with the glycosylation that the being seen O-of Pichia pastoris cell of the deletion that contains one of PMT1 or PMT2 gene is connected, produced the recombinant protein of the level of glycosylation of the O-connection with reduction.Above-mentioned Pmti inhibition makes to produce the glycosylation quantity that is connected with O-on the protein that produces in the similar host cell culture that lacks the Pmti inhibition with about 0.03 μ M to the quantity of 20 μ M and compares, has the glycosylated protein that the O-of reduction connects.Result displayed has shown that further the host cell culture can grow under the situation that has some amount Pmti inhibition in embodiment 3, and described quantity is enough to suppress the glycosylation that O-connects and does not kill described host cell.

Described method can comprise in the substratum that contains one or more Pmt inhibitions adds one or more α-1, and the 2-mannosidase produces the glycosylated recombinant protein of the O-connection with reduction.α-1,2-mannosidase are the conservative families for the eucaryon enzyme of the maturation of N-glycan, and it can be with Man in yeast ₉GlcNAc ₂Be trimmed to Man ₈GlcNAc ₂(Vallee et al.，2000，EMBO J.，19：581-588)。α-1,2-mannosidase are also referred to as I class a-mannosidase, and it has identified (Kawar et al., 2000, Glycobiology 10:347-355) in Mammals, eucaryon species such as low and insect cell.Mammalian cell is known to have several I class alpha-Mannosidases, some of them can be repaired multiple mannose residue (Moremen et al., 1994, Glycobiology 4:113-125), and yeast seems to have α-1 still less, more special, the 2-mannosidase.For example, disclose Saccharomyces and had the single α-1 that is encoded by MNN1, the 2-mannosidase, it removes specific mannose residue (for example a, Man ₉GlcNAc ₂To Man ₈GlcNAc ₂) (Herscovics, 1999, Biochim Biophys Acta., 1473:96-107).Thereby, for example there is and can not removes from glycan structures the endogenous α-1 of a plurality of mannose residues in fungi and the yeast at many lower eukaryotes, the 2-mannosidase can not allow to have the generation of the glycosylated protein that the O-of reduction connects.Therefore, method herein need be introduced the α-1 that can repair a plurality of mannose residues from the glycan that O-connects in the substratum that contains host cell, the 2-mannosidase, or introduce coding in the host cell can be from the rest and reorganize α-1 of a plurality of mannose residues of the glycan that O-connects, the nucleic acid of 2-mannosidase.α herein-1,2-mannosidase comprise complete, natural α-1,2-mannosidase; Modified to improve its α-1, the α-1 of 2-mannoside enzymic activity, 2-mannosidase; Modified to reduce its α-1, the α-1 of 2-mannoside enzymic activity, 2-mannosidase; And, at least comprise and have α-1, the recombinant alpha-1 of the catalyst structure domain of 2-mannoside enzymic activity, the 2-mannosidase is (for example, comprise and have α-1, the catalyst structure domain of 2-mannoside enzymic activity, be fused to the fusion rotein of heterologous protein, polypeptide or peptide).

In specific embodiment, the glycan a plurality of mannose residues of finishing that can connect from O-also are added to α-1 cell culture, and the 2-mannosidase is produced by Trichoderma sp., Saccharomyces sp. or Aspergillus sp..Current, preferred α-1, the 2-mannosidase is available from Trichoderma reesei, Aspergillus niger or Aspergillus oryzae.T.reesei is also referred to as Hypocrea jecorina.In embodiment 3, comprise express recombinant α-1, the yeast of the conversion of the expression cassette of 2-mannosidase is used to produce the glycosylated recombinant protein of the O-connection with reduction, described recombinant alpha-1, the 2-mannosidase comprises the Trichodermareesei α-1 that merges with Saccharomycescerevisiea α MAT front signal sequence (pre signal sequence), 2-mannosidase catalyst structure domain.Can be used for the recombinant alpha-1 that method herein produces the glycosylated protein of the O-connection with reduction, another example of 2-mannosidase is at Maras et al., 2000, disclosed reorganization Trichoderma reesei α-1 among the J.Biotechnol.77:255-263, the 2-mannosidase, Trichoderma reesei α-1 wherein, 2-mannosidase catalyst structure domain are fused to original signal peptide (prepro-signal peptide) before Saccharomyces cerevisiea α-MAT.

α-1, the 2-mannosidase also can produce from chimeric nucleic acid, described chimeric nucleic acid comprises at least the α-1 of a plurality of mannose residues of glycan finishing that coding can connect from O-, the catalyst structure domain of 2-mannosidase, the nucleotide sequence that is operatively connected with the nucleotide sequence of common not relevant with the described catalyst structure domain cell-targeting signal peptide of coding.Described chimeric nucleic acid can be operably connected with constitutive promoter or inducible promoters.Chimeric nucleic acid is transformed into and produces α-1 in the host cell, the 2-mannosidase, and it is separated and add in the expression of heterologous protein is induced in the cell culture medium that contains cell then, and described cell transforms with the nucleic acid of coding heterologous protein.Alternatively, with coding for alpha-1, the nucleic acid transformed host cell of the chimeric nucleic acid of 2-mannosidase and coding recombinant protein, while coexpression described α-1,2-mannosidase and recombinant protein.In specific embodiment, limit α-1, the nucleic acid of the chimeric nucleic acid of 2-mannosidase and coding recombinant protein all is operably connected with inducible promoters.In other embodiments, one of described promotor or both are composing types.Embodiment 3 has illustrated described method, the nucleic acid of alpha-Mannosidase and recombinant protein of wherein encoding is operably connected to constitutive promoter, be imported in the host cell, the culture of hatching host cell then under the situation that has one or more Pmt inhibitions produces the glycosylated recombinant protein of the O-connection with reduction.Embodiment 3 has shown that further Pmt1 inhibition and α-1, and the 2-mannosidase is fit to reduce synergistically the glycosylated quantity that O-connects, and compares with the glycosylated quantity that O-under the situation that has independent any is connected.

Aspect specific, the glycosylation that the O-of reduction connects can be by only adding one or more α-1 to substratum, and 2-mannosidase but not one or more Pmt inhibitions are realized.In one aspect, the nucleic acid of coding recombinant protein is operably connected with inducible promoters, and it allows that being expressed in when expecting of described recombinant protein is induced.In yet another aspect, the nucleic acid of coded protein is operably connected with constitutive promoter.For the ease of the separation of the recombinant protein of expressing, preferably current, described protein comprises signal sequence, and described signal sequence instructs recombinant protein to be secreted in the cell culture medium, thereby recombinant protein can be separated therein.

Aspect first, adding one or more α-1 to substratum, before the 2-mannosidase, transformed host cells is cultivated a large amount of host cells that time enough produces the expectation of the recombinant protein that is enough to produce desired amt.Inductor and one or more α-1, the 2-mannosidase can side by side add in the culture, perhaps adding one or more α-1, add inductor to culture before the 2-mannosidase, perhaps before adding inductor, add one or more α-1 to culture, the 2-mannosidase.The glycosylated recombinant protein of inducing that generation has the O-connection of reduction can reclaim from substratum, perhaps for the protein that does not have signal sequence, reclaims from host cell by cracking.

Aspect second, the nucleic acid of recombinant protein of wherein encoding is operably connected with constitutive promoter, when being established, culture in substratum, adds one or more α-1, the 2-mannosidase, the glycosylated recombinant protein that the O-with reduction that produces connects can reclaim from substratum, or for the recombinant protein that does not have signal sequence, reclaim from host cell by cracking.

The glycosylated inhibition production that connects at the O-that does not use Pmt mediation have glycosylated protein that the O-of reduction connects more further aspect, with coding for alpha-1, the nucleic acid transformed host cell of the chimeric nucleic acid of 2-mannosidase and coding recombinant protein, coexpression α-1,2-mannosidase and described recombinant protein produce the glycosylated recombinant protein that the O-of reduction is connected.In specific embodiment, coding for alpha-1, the nucleic acid of the chimeric nucleic acid of 2-mannosidase and coding recombinant protein all is operably connected with inducible promoters.In other embodiments, one of described promotor or both are composing types.For inducible promoters, inducing α-1, the host cell of growing before the expression of 2-mannosidase and/or recombinant protein produces a large amount of host cells of expectation.Embodiment 3 has illustrated described method, coding for alpha-1 wherein, the nucleic acid of 2-mannosidase and recombinant protein is operably connected with constitutive promoter, be imported in the host cell, the culture certain hour of hatching described host cell then produces recombinant protein, with lacking α-1, the recombinant protein that produces in the cell of 2-mannosidase is compared, described recombinant protein has the glycosylation that the O-of reduction connects.

II. host cell

Comprise higher eukaryotic cell and lower eukaryotes cell though be used for the host cell of method herein, for example filamentous fungus or yeast cell are current is preferred for protein expression for the lower eukaryotes cell, because they can be cultivated economically, obtain the protein of high yield, when suitably modifying, can produce the protein with suitable glycosylation pattern.The lower eukaryotes cell (for example comprises yeast, fungi, ring-flagellate, microsporozoite, alveolates, dinoflagellate), stramenopiles (for example, brown alga, protozoon), rhodophyta (for example, red algae), plant (for example, green alga, vegetable cell, liver moss) and other protobionts.Yeast and fungi include but not limited to: Pichiasp. (Pichia pastoris for example, Pichia finlandica, Pichia trehalophila, Pichiakoclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia tkermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica), Saccharomyces sp. (f is Saccharomyces cerevisiea for example), Hansenulapolymorpha, Kluyveromyces sp. (for example, Kluyveromyces lactis), Candidaalbicans, Aspergillus sp (for example, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae), Trichoderma reesei, Chrysosporium lucbiowense, Fusarium sp. (for example, Fusarium gramineum, Fusarium venenatum), Physcomitrella patens and Neurospora crassa.Especially, yeast is current preferred, because yeast provides the genetics of determining, allows the protein positioning strategy of quick conversion, test and is convenient to the gene knockout technology.The carrier that is fit to has expression control sequenc, and for example promotor comprises 2-phosphoglyceric acid kinases or other glycolytic ferments, replication orgin, terminator sequence, etc., as expected.

Each primary yeast, for example K.lactis, Pichia pastoris, Pichia methanolica and Hansenula polymorpha are current preferred for cell cultures, because they can grow into a large amount of recombinant protein of high-cell density justacrine.Similarly, filamentous fungus, for example Aspergillus niger, Fusarium sp, Neurospora crass etc. can be used for producing recombinant protein with technical scale.

Lower eukaryotes, particularly filamentous fungus and yeast, being modified hereditarily, thus their marking proteins or glycoprotein, wherein glycosylation pattern is anthropoid or humanized.This can realize by eliminating selected endogenous glycosylase and/or supplemented with exogenous enzyme, as Gerngross etc. in U.S. Patent No. US7029872 and described in the disclosed patent application Nos.20040018590 of the U.S., 20050170452,20050260729,20040230042,20050208617,20040171826,20050208617,20060160179,20060040353 and 20060211085.Thereby host cell can additionally or selectively be expressed one or more enzymes or enzymic activity by through engineering approaches, and its permission produces specific N-glycan structures with high yield.Such enzyme can be by target host subcellular organelle, and described enzyme will have optimum activity therein, for example, and by the mode of not relevant with described enzyme usually signal peptide.Host cell also can be modified to express sugar nucleotide translocator and/or nucleotide pyrophosphatase.Translocator and diphosphatase have improved the effectiveness of the glycosylation step of through engineering approaches, by the suitable substrates that glycosylase is provided in suitable compartment, the elimination that reduces competitive product inhibition and promote nucleoside diphosphate.Referring to, for example disclosed patent application No.20040018590 of the U.S. of Gerngross et al. and Hamilton, 2003, Science 301:1244-46 and above-mentioned United States Patent (USP) and patent application.

For instance, host cell (for example, yeast or fungi) can be selected or through engineering approaches exhausts 1,6-mannose transferase activity, it will add mannose residue in addition on the N-of glycoprotein glycan, and further comprising nucleic acid for the ectopic expression of α-1,2 mannoside enzymic activity, it allows to produce the Man that has greater than 30 molar percentages ₅GlcNAc ₂The recombinant glycoprotein of N-glycan.When producing glycoprotein according to method described here in host cell, described host cell will produce mainly has Man ₅GlcNAc ₂N-glycan structures structure and the glycosylated glycoprotein of O-of comparing reduction with the glycoprotein that in other cell, produces.Further, further to comprise the nucleic acid for the ectopic expression of GlcNAc transferase I activity, it allows to produce mainly has GlcNAcMan to described host cell by through engineering approaches ₅GlcNAc ₂The glycoprotein of N-glycan structures.When producing glycoprotein according to method described here in host cell, described host cell will produce mainly has GlcNAcMan ₅GlcNAc ₂N-glycan structures and the glycosylated glycoprotein of O-of comparing reduction with the glycoprotein that in other cell, produces.More further aspect, further to comprise the nucleic acid for the ectopic expression of mannosidase II activity, it allows to produce mainly has GlcNAcMan to described host cell by through engineering approaches ₃GlcNAc ₂The glycoprotein of N-glycan structures.

When producing glycoprotein according to method described here in host cell, described host cell will produce mainly has GlcNAcMan ₃GlcNAc ₂N-glycan structures and the glycosylated glycoprotein of O-of comparing reduction with the glycoprotein that in other cell, produces.More further aspect, further to comprise the nucleic acid for the ectopic expression of GlcNAc transferase I I activity, it allows to produce mainly has GlcNAc to described host cell by through engineering approaches ₂Man ₃GlcNAc ₂The glycoprotein of N-glycan structures.When producing glycoprotein according to method described here in host cell, described host cell will produce mainly has GlcNAc ₂Man ₃GlcNAc ₂N-glycan structures and the glycosylated glycoprotein of O-of comparing reduction with the glycoprotein that in other cell, produces.More further aspect; above-mentioned host cell can be produced specific hybridization or compound N-glycan or human sample N-glycan structures by further through engineering approaches; relate to glycosylated one or more higher eucaryote genes that N-connects by further comprising with arbitrary combination, described genes encoding is saliva acyltransferase activity, II class and III class mannoside enzymic activity, GlcNAc transferase I I, III, IV, V, VI, IX activity and galactotransferase activity for example.Preferably current, described cell further comprises one or more nucleic acid of coding UDP specificity diphosphatase activity, GDP specificity diphosphatase activity and UDP-GlcNAc translocator activity.

Plant and plant cell cultures can be used for being expressed in glycosylated protein that the O-with reduction of this instruction is connected and glycoprotein (referring to, for example, Larrick ﹠amp; Fry, 1991, Hum.Antibodies Hybridomas 2:172-89); Benvenuto et al., 1991, Plant Mol.Biol.17:865-74); Durin et al., 1990, Plant Mol.Biol.15:281-93); Hiatt et al., 1989, Nature 342:76-8).Preferably plant host comprises, for example, and Arabidopsis, Nicotiana tabacum, Nicotiana rustica and Solarium tuberosum.

Glycosylated glycoprotein protein and glycoprotein that insect cell culture also can connect for the production of the O-with reduction in this instruction, for example based on the expression system of baculovirus (referring to, for example, Putlitz et al., 1990, Bio/Technology 8:651-654).

Though current cultivation economically unlike lower eukaryotes and prokaryotic organism, the mammalian tissues cell culture also can be used for be expressed the glycosylated protein that is connected with the O-with reduction that is created in this instruction and glycoprotein (referring to Winnacker, From Genes to Clones (VCHPublishers, NY, 1987).The host who is fit to comprises Chinese hamster ovary celI system, various COS clone, HeLa cell, preferred myeloma cell line etc., or the B cell or the hybridoma that transform.The expression vector of these cells can comprise expression control sequenc, for example replication orgin, promotor, enhanser (Queen et al., 1986I, mmunol.Rev.89:49-68) and essential machining information site, for example ribosome bind site, RNA splice site, polyadenylic acid site and Transcription Termination subsequence.Expression control sequenc is the promotor from immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus etc.Usually, but selective marker, and for example the neoR expression cassette is included in the expression vector.

Coding wants the nucleic acid of expressed protein to transfer in the host cell by ordinary method, and it depends on the kind of cell host and becomes.For example, calcium phosphate is handled, protoplastis merges, hatch naturally, fat transfection, particle gun, can be used for cell host based on transduction or the electroporation of virus.Tungsten particle bullet transgenosis is preferred for vegetable cell and tissue.(usually referring to Maniatiset al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Press, 1982)).

In case expressed, glycosylated protein or glycoprotein with O-connection of reduction can come purifying according to the standard method of this area, comprise ammonium sulfate precipitation, affinity column, column chromatography, gel electrophoresis etc.(usually, referring to, Scopes, R., Protein Purification (Springer-Verlag, N.Y., 1982)).For pharmaceutical use, be preferred at least about 90 to 95% homogeneous pure glycoprotein basically, 98 to 99% or more homogeneous be most preferred.In case be purified, purifying or be purified to homogeneous partly as expected, described protein can use (comprising external ground (extracorporeally)) remedially, or in exploitation or carry out using aspect analytical procedure, immunofluorescence technique dyeing etc.(usually referring to, Immunological Methods, and Vols.I and II (Lefkovitsand Pernis, eds., Academic Press, NY, 1979and 1981).

Therefore, further provide glycoprotein compositions, it comprises the N-glycan structures of main kind, and with the glycosylated inhibition or the α-1 that are not connected at the O-of Pmt mediation, the composition of the glycoprotein that produces in the host cell of hatching under 2-mannosidase or the two the situation is compared, glycosylation with O-connection of reduction, described α-1,2-mannoside endonuclease capable surpasses a mannose residue from the glycan structures finishing.Aspect specific, described glycoprotein compositions comprises having and is selected from by Man ₅GlcNAc ₂, Man ₃GlcNAc ₂, GlcNAcMan ₅GlcNAc ₂, GlcNAcMan ₃GlcNAc ₂, GlcNAc ₂Man ₃GlcNAc ₂, GalGlcNAcMan ₅GlcNAc ₂, Gal (GlcNAc) ₂Man ₅GlcNAc ₂, (GalGlcNAc) ₂Man ₅GlcNAc ₂, NANAGalGlcNAcMan ₃GlcNAc ₂, NANA ₂Gal ₂GlcNAcMan ₃GlcNAc ₂And GalGlcNAcMan ₃GlcNAc ₂The glycoprotein of the main N-glycan structures of sugar form.

III. pharmaceutical composition

Glycosylated protein and glycoprotein with O-connection of reduction can be impregnated in the pharmaceutical composition, described pharmaceutical composition comprise glycoprotein as active treatment reagent and multiple other pharmaceutically acceptable compositions (referring to Remington ' s Pharmaceutical Science (15th ed., MackPublishing Company, Easton, Pennsylvania, 1980).Preferred form depends on the expection mode of using and treating application.The preparation that depends on expectation, composition also can comprise pharmaceutically acceptable, nontoxic carrier or thinner, and it is defined as being generally used for preparing the vehicle of the pharmaceutical composition of using for animal or human's class.Thereby select thinner not influence the biologic activity of combination.The example of these thinners is distilled water, physiology phosphate buffered saline (PBS), Ringer ' s solution, glucose solution and Hank ' s solution.In addition, pharmaceutical composition or preparation also can comprise other carriers, adjuvant, or the stablizer of nontoxic, non-therapeutic, non-immunogenic, etc.

The pharmaceutical composition that is used for parenteral administration is aseptic, first-class substantially that ooze, pyrogen-free, and according to FDA or similar means GMP prepare.Glycoprotein can be used as the solution of material in physiology ground acceptable diluent or the injectable dosage of suspension is used, and having can be for example pharmaceutical carrier of water, oil, salt solution, glycerine or ethanol of sterile liquid.In addition, the auxiliary material, for example wetting agent or emulsifying agent, tensio-active agent, pH value buffer reagent etc. may reside in the composition.Other parts of pharmaceutical composition are those of oil, animal, plant or synthetic source, for example, and peanut oil, soya-bean oil and mineral oil.Usually, ethylene glycol for example propylene glycol or polyoxyethylene glycol is preferred liquid vehicle, particularly for Injectable solution.Glycoprotein can be used with the form of bank injection or implant goods, and it can be prepared in the mode of the lasting release that allows activeconstituents.Usually, this composition is prepared as injectable, as liquor or suspension; Also can be prepared as the solid form that before injection, is suitable for being dissolved in or being suspended in liquid vehicle.Goods can also emulsification be sealed in liposome or particulate in, for example polylactide, poly-glycollide or multipolymer are used for the adjuvant effect that strengthens, and be as discussed above.(referring to Langer, Science 249,1527 (1990) and Hanes, Advanced DrugDelivery Reviews 28,97-119 (1997).

Unless in this other definition, the Science and Technology term of related use with the present invention and term have the implication of those of ordinary skills' common sense.Further, unless context needs in addition, the term of odd number should comprise plural number, and the term of plural number should comprise odd number.Usually, the relevant name of using, and the technology of biological chemistry described here, zymetology, molecule and cytobiology, microbiology, genetics and protein and nucleic acid chemistry and hybridization is known in this field and normally used.According to ordinary method well known in the art and as various and the description of reference more specifically usually carry out method of the present invention and technology, these reference are cited in this manual and discuss unless otherwise stated.Referring to, for example, Sambrook et al.Molecular Cloning:A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates (1992, andSupplements to 2002); Harlow and Lane, Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ.Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, NJ; Handbook of Biochemistry:Section A Proteins, Vol I, CRCPress (1976); Handbook of Biochemistry:Section A Proteins, Vol n, CRCPress (1976); Essentials of Glycobiology, Cold Spring Harbor LaboratoryPress (1999).

Following examples intention promotes further understanding of the present invention.

Embodiment 1

This embodiment provides the method for preparing various Pt inhibitions.Unless other regulation, (St.Louis is MO) and by the use of receiving available from Sigma-Aldrich Chemical Co. for all materials.All intermediates and final product ¹H NMR spectrum and disclosed data consistent.

Pmti-1 (5-[[3,4-two (phenyl methoxyl group) phenyl] methylene radical]-4-oxo-2-sulfo--3 thiazolidine acetate) preparation, as follows.

Operation is adapted in people's U.S. Patent No.s such as Orchard 7,105,554.Rhodanine-3-acetic acid (1g, 5.20mmol, 1eq.), 3,4-benzyloxy phenyl aldehyde (2.04g, 6.25mmol, 1.2eq.) and sodium-acetate (3eq.) solution in acetic acid (30mL) is heated to backflow for 1.3g, 15.6mmol, and stirring is spent the night.Along with the reaction mixture cool to room temperature, product is precipitated, filter, and with acetic acid, use petroleum ether then.Resistates is dissolved among the hot DMSO, filters, and precipitates by adding water.In cooling, throw out be filtered and from ethyl acetate and sherwood oil recrystallize, obtain product, product be suspended in the water and in a vacuum freeze-drying after the final product that obtains fluffy yellow powder yesterday.

Pmti-2,2 (5-[[3-(1-phenyl ethoxy)-4-(2-phenyl ethoxy)] phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate) be prepared as follows.

According to Orchard etc. in U.S. Patent No. 7,105, the synthetic this product of guidance in 554.Rhodanine-3-acetic acid (375mg, 1.96mmol, 1eq.), 3-(1-phenyl ethoxy)-4-(2-phenyl ethoxy) phenyl aldehyde (680mg, 1.96mmol, 1eq.) and ammonium acetate (453mg, 3eq.) solution be heated to 70 ℃ ten minutes, then cool to room temperature and with ethyl acetate (100mL) dilution.Organic solution with 1M HCl (2 * 200mL) and salt solution (200mL) washing, dry and evaporate on sodium sulfate then.35-75 μ m C18 (Alltech Associates, Deerfield, the liquid chromatography purified product of 10 * 2.5cm glass column IL) have been filled in use.Adopt gradient elution.Buffer A is 0.1% acetic acid, and buffer B is 80% acetonitrile.Gradient comprise 20%B three minutes, in 40 minutes, bring up to 75%B.Flow velocity is 8mL/ minute.Detection is carried out at 280nm.Suitable fraction is merged, and concentrate, and freeze-drying obtains the product of fluffy yellow powder in a vacuum.

Pmti-3, (5-[[3-(1-phenyl-2-hydroxyl) oxyethyl group)-4-(2-phenyl ethoxy)] phenyl] methylene radical]-4-oxo-2-sulfo--3-thiazolidine acetate), the goods of (Orchard et al. U.S. Patent No. 7,105,554) are synthetic in following three steps.

Step 1:(+)-(S)-generation of 2-acetoxyl-1-bromo-1-phenylethane.Cold HBr-acetic acid in about five minutes (12.4g 52.2mmol) is added drop-wise to (-)-(R)-1-phenylethane-1, the 2-glycol (2.4g, 17.4mmol) in, mixture at room temperature stirred 40 minutes.Add water (25mL), with yellow soda ash this solution that neutralizes, (3 * 30mL) extract with ether.The extract of combination is dried and evaporates to obtain (+)-(S)-2-acetoxyl-1-bromo-1-phenylethane (3.93mg, 93%), (d25 1.415g/mL, [x] ₀24+93.5 ° (CCl ₄In c 5.63) 2.72 (and 5H, s), 4.98 (aH, dd, 6.7 and 7.0Hz) and 5.56 (2H, d).This product is not distilled.Determine the isometry uniformity by comparing nmr spectrum (not having PhCH*OAc resonance) with 1,2-diacetoxyl-1-phenylethane.(noticing that racemic reagent is substituted by listed optical isomer).

Step 2:3-[(1-phenyl-2-hydroxyl) oxyethyl group]-production of 4-(2-phenyl ethoxy)-phenyl aldehyde.(2-acetoxyl-1-bromotrifluoromethane) benzene (3.32g, 13.67mmol, 1.2eq) (product of step 1) add 3-hydroxyl-4-(2-phenyl ethoxy)-phenyl aldehyde (2.76g under stirring, 11.39mmol, 1eq.) and cesium carbonate (2.97g, 9.11mmol, 0.8eq.) at N, in the solution in the dinethylformamide (15mL).Solution at room temperature stirred 19 hours, stirred 21 hours at 80 ℃ then.Start reaction (add salt solution and help destroy formed emulsion) by between ethyl acetate and water, distributing.Water, salt water washing organic layer twice again, dry and evaporation obtains dark oil on sodium sulfate then.Resistates obtains orange oil by chromatography on silica gel with Anaesthetie Ether wash-out purifying.This oil is dissolved in the methyl alcohol (100ml), to this solution add sodium hydroxide the aqueous solution (7mL, 1M).After 30 minutes, mixture is evaporated (to remove methyl alcohol), distributes resistates between methylene dichloride and water.Organic layer is dry and evaporation on sodium sulfate.Resistates is used sherwood oil by chromatography purifying on silica gel: Anaesthetie Ether (1: 2) wash-out, obtaining product is cream-coloured powder.

The generation of step 3:Pmti-3.Rhodanine-3-acetic acid (158mg, 0.828mmol, 1eq.), 3-(1-phenyl-2-hydroxyl) oxyethyl group)-4-(2-phenyl ethoxy) phenyl aldehyde (300mg, 0.828mmol, 1eq.) (product of step 2) and ammonium acetate (191mg, 3eq) solution in toluene (10mL) is heated to and refluxed 3.5 hours, and cool to room temperature is with ethyl acetate (50mL) dilution.Organic solution with 1MHCl (2 * 200mL) and salt solution (200mL) washing, dry and evaporate on sodium sulfate then.After handling, resistates is by chromatography purifying on silica gel.Use eluent ethyl acetate to obtain yellow glue, its recrystallize from Anaesthetie Ether and sherwood oil obtains the product of yellow powder.

Embodiment 2

This embodiment has shown that the expression vector with coding kringle 1-3 mark glycoprotein transforms and have with the Pichia pastcris generation that the Pmt inhibition is handled the glycosylated glycoprotein of O-of reduction.

The plasmid DNA of the report glycoprotein of coding His label is transformed into and produces bacterial strain yJC53 among the wild-type Pichiapastoris, and described plasmid DNA is made of human plasminogen structural domain K1, the K2 and the K3 (Kringle 1-3 protein) that are under the control of Pichia pastoris alcohol oxidase 1 (AOX1) promotor.The Kringle reporter protein matter that is made of structural domain K1, K2, K3 and K4 is at Duman et al.Biotechnol.Appl.Biochem. (1998), v.28, p.39-45and only domain K3 in Choi et al., 2003, Proc.Natl.Acad.Sci.U.S.A.100 (9): discussed among the 5022-5027.The aminoacid sequence of the Kringle 1-3 protein of Shi Yonging is in this embodiment

SECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPE KRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRE LRPWCFTTDPNKRWELCDIPRCTT PPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHSRT PENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQ LAPTAPPELTPVVQDGGGH HHHHHHHH(SEQ ID NO：1).

Kringle 1-3 albumen contains at least two potential Mammals O-glycosylation sites that meet the consensus sequence P that claims-1 and+3: the glycosylated serine residue of O-, in aminoacid sequence " PPPSsgp ", capitalize, and the glycosylated threonine residues of O-, in aminoacid sequence " lapTapp ", capitalize.The O-glycosylation site is underscore in above aminoacid sequence.Potential Mammals O-glycosylation site is between K1 and K2 structural domain and K2 and K3 structural domain.Yet as shown in the table 1, in yeast, protein has the glycosylation site that about 20 O-connect.Thereby under the glycosylated situation by the O-of inhibition shown in method herein connection, the glycosylation that O-connects may not be the remarkable shortcoming of producing protein in yeast.The N-glycosylation site is present in the K3 structural domain, and it is removed by the l-asparagine of replacing 208 places, position of SEQ ID NO:1 with Serine.Therefore, only glycan will be the glycosylated result of O-on Kringle 1-3 protein.

The plasmid that contains encoded K ringle 1-3 protein DNA uses forward primer K1-3/UP5 '-CGGAA TTCTC AGAGT GCAAG ACTGG GAATA GAA-3 ' (SEQ IDNO:2) and reverse primer K1-3/LP1 (reverse primer, 3Gly+2His, with K1-3/UP pairing) 5 '-ATGAT GATGA CCACC ACCGT CCTGG ACCAC AGGGGTTAG-3 ' (SEQ ID NO:3) preparation produces the PCR product, it uses reverse primer K1-3/LP2 (reverse primer then, the 3Gly+9His+ terminator codon is with the K1-3/UP pairing) 5 '-TTAATGATGA TGATG ATGAT GATGA TGATG ACCAC CACC-3 ' (SEQ IDNO:4) pcr amplification.The PCR condition is as follows: after 95 ℃ of 1 circulations in 2 minutes as denaturing step, 95 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 30 circulations of 1 minute, 72 ℃ of 1 circulations of 10 minutes are then carried out in the PCR reaction.After the column purification of PCR reaction and PCR product, the Nucleotide A projection of PCR product uses ExTaq to produce (72 ℃ of 1 circulations in 15 minutes).The PCR product that produces is used for second PCR reaction as pcr template, primer K1-3/UP and the K1-3/LP2 wild-type Kringle 1-3+3Gly+9His that is used to increase wherein, and it is cloned in the pCR2.1 plasmid vector (Invitrogen) and produces pBK105.Following PCR primer suddenlys change from aminoacid sequence SRTP generation aminoacid sequence NRTP to Ser for generation of the Asn at 208 places, position in the Kringle 1-3 protein then: forward primer K3f (Asn is to Ser) 5 '-ACCCCTCACACACATTCTAGGACACCAGAAAACTTC-3 ' (SEQ ID NO:5) and reverse primer K3r 5 '-CTGTGCACTCCAGTGCTGACAGGTGTG-3 ' (SEQ ID NO:6).Producing Asn by inverse PCR in pBK105 then suddenlys change to Ser.The PCR condition is as follows: after 95 ℃ of 1 circulations in 2 minutes as denaturing step, 95 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 30 circulations of 5 minutes, 72 ℃ of 1 circulations of 10 minutes are then carried out in the PCR reaction.Connect the PCR product that produces and come production of plasmid pBK118, it is checked order to confirm sudden change.

Plasmid pBK118 digests with EcoRI, and dna fragmentation is by gel-purified and be cloned into pPICZaA (EcoRI site CA) produces pBK119 (Pichia expression plasmid) for Invitrogen, La Jolla.Plasmid pPICZaA contains α-factor secretion signal, and it allows that most protein is from the effective secretion of Pichia pastoris; 5 '-AOX, contain the 942bp fragment of AOX1 promotor, but it allows methanol induction and high-caliber expression among the Pichia pastoris; And the ZEOCIN resistant gene is used for the just selection at E.coli and Pichia pastoris.Before in being transformed into Pichia pastoris bacterial strain, plasmid pBK119 PmeI linearizing.Plasmid pBK is transformed in Pichia pastoris bacterial strain yJC53, wild type strain and the various PMT knock-out bacterial strain.

PMT knocks out yeast strain according to Gentzsch and Tanner, and EMBO J.1996Nov1; 15 (21): among the 25752-5759 for Saccharomyces cerevisiae listed operating among the Pichia pastoris create.The nucleotide sequence that Pichia pastoris PMT gene utilizes Saccharomycescerevisiae PMT gene by homology search available from IntegratedGenomics, Chicago identifies in the genomic nucleotide sequence of Pichia pastoris of IL.The deletion of Pichia pastoris PMT (PpPMT) gene is as follows.By the PCR method of superposition produce PpPMT deletion allelotrope (referring to, Davidson et al. for example, 2004, Glycobiology14:399-407; Ho et al., 1989, Gene 77:51-9; Horton et al., 1989, Gene77:61-8).In PCR reaction, comprise 5 of PMT gene ' and 3 ' flank region and NAT or HYG resistance markers (Goldstein and McCusker, 1999, Yeast 14:1541-1553; The DNA of nucleotide sequence Goldstein et al., 1999, Yeast 15:507-110) is by pcr amplification.Flank uses available from Integrated Genomics in the primer sequence in the zone of PMT gene, Chicago, and the Pichia pastoris genome nucleotide sequence of IL designs as instructing.The Pichiapastoris genomic dna is used as the template of PpPMT flank region pcr amplification, and NAT and HYG fragment are used (Goldstein, and McCusker, 1999, ibid.; Goldstein et al., 1999, ibid.) the middle plasmid of describing carries out pcr amplification as template.Then, in the 2nd PCR reaction, all three kinds of first round PCR products be used as template produce contain all three kinds of fragments overlapping product as single linear allelotrope.Directly adopt final PCR product to be used for transforming then.Select transformant at the YPD substratum that contains 200 μ g/mL homomycin or 100 μ g/mL nourseothricins.In each case, confirm the correct integration of mutant allele by PCR.The PMT knock-out bacterial strain of creating be yJC51 (the pmt3 Δ, the pmt5 Δ, the pmt6 Δ), yJC55 (pmt1 Δ), yJC66 (pmt2 Δ), and yJC65 (pmt4 Δ).The plasmid pBK119 of the above-mentioned Kringle 1-3 protein of each personal coding of PMT knock-out bacterial strain transforms.

The Kringle 1-3 protein expression of the yeast strain that transforms uses the glycerine-mixture substratum (BMGY) of buffering to carry out at 24 ℃ in shaking bottle, and described substratum is by 1% yeast extract, 2% peptone, 100mM vitriolate of tartar pH of buffer 6.0,1.34% yeast nitrogen, 4 * 10 ^-5% vitamin H and 1% glycerine are formed.The inducing culture that is used for protein expression is the methyl alcohol-mixture substratum (BMMY) of buffering, and it replaces glycerine to form by 1% methyl alcohol among the BMGY.Pmt inhibition Pmti-1, Pmit-2 in the methyl alcohol or Pmti-3 add the final concentration that arrives 0.2 μ M, 2 μ M or 20 μ M in the growth medium to, add inducing culture this moment.Harvested cell, at 2,000rpm centrifugal five minutes.Pmt inhibition Pmti-1, Pmti-2 and Pmti-3 are interchangeable basically, have very little variation at the easness that uses.For example, under described cell culture condition, the solubleness of Pmti-3 is greater than Pmti-1 and Pmti-2, thereby is expectation among the three.

The yJC53 culture that the Pmti-1 that uses by oneself handles or the 7 μ L supernatant liquors of yJC55 are according to Laemmli, U.K. (1970) Nature 227,680-685 separates by polyacrylamide gel electrophoresis (SDS-PAGE), then at nitrocellulose membrane (Schleicher ﹠amp; (Florham Park NJ) goes up electroblotting to Schuell for present Whatman, Inc..Kringle 1-3 protein uses from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) anti-His antibody (H-15) detects at the Western trace, use ImmuunoPure Metal Enhanced DAB SubstrateKit (Pierce Biotechnology, Rockford, IL) colour developing.As shown in the swimming lane 1 of the Western trace shown in the accompanying drawing 1, from the Kringle 1-3 protein of untreated Pichia pastoris since the glycosylated existence of O-run and be disperse.Yet, by contrast, from the Kringle1-3 protein of yJC53 (the Pichia pastoris that handles with 2 or 20 μ M Pmti-1, be respectively swimming lane 2 and 3) represented different bands owing to lack the O-glycosylation, be similar to the Kringle 1-3 (the pmt1 Δ of Pichia pastoris knocks out mutant) (swimming lane 4 and 5) that expresses from yJC55.Accompanying drawing 1 has shown that further the O-glycosylation that Pmti-1 reduces reaches viewed similar level in the bacterial strain that lacks Pmt1.

For the O-glycosylation of measuring the Pmt inhibition reduces, use nickel chelating chromatography from growth medium purifying Kringle 1-3 protein (Choi et al., 2003, Proc.Natl.Acad.Sci.U.S.A.100 (9): 5022-5027), eliminate (β-elimination) (Harvey by alkali, 1999 MassSpectrometry Reviews 18 349-451) discharge and separate the O-glycan from Krinngle 1-3 protein.This processing has also reduced the reduction end of the new formation of the O-glycan (few seminose or seminose) that N.F,USP MANNITOL is discharged.N.F,USP MANNITOL group thereby served as the indicator of the uniqueness of each O-glycan.The 0.5nmole that contains in 100 μ L volume PBS damping fluids or more Kringle 1-3 protein are that the β elimination is required.Sample was hatched 16 hours at 50 ℃ with 25 μ L alkaline hydrogen boride agent treated.Add about 20 μ L arabitol internal standards, add 10 μ L Glacial acetic acid subsequently.Sample is the centrifugal Millipore strainer that contains SEPABEADS and AG 50W-X8 resin and water flushing that passes then.Sample comprises washings, transfers in the plastics automatic sampler bottle, is evaporated to drying in centrifugal evaporator.150 μ l 1%AcOH/MeOH add in the sample, and sample is evaporated to drying in centrifugal evaporator.This last step repeats five times again.Add 200 μ L water, be accompanied by pulse electrochemical detection-DionexHPLC (HPAEC-PAD) by high pH anion-exchange chromatography and analyze 100 μ L samples.The average O-glycan of takeoff according to the N.F,USP MANNITOL that reclaims takies.The result summarizes in table 1, it has shown that in the Pichia pastoris bacterial strain that contains complete PMT1 and PMT2 gene any Pmt Chemical Inhibition thing is reduced to certain level with the glycosylation that the O-of the Kringle 1-3 protein of secretion is connected, and described level is comparable with the glycosylated level that the being seen O-of cell of the deletion that contains PMT1 or PMT2 gene is connected.Though table 1 has also shown this protein and has had the glycosylation site that two potential Mammals O-connect that this protein has kept the glycosylation site that about 20 O-connect in yeast.

Table 1

Embodiment 3

In this embodiment, caused the generation of the glycosylated protein of O-with reduction with the DNA transformed yeast cells of coding T.reesei alpha-Mannosidase, the O-glycosylation is further reduced when hatching under also there is the situation of Pmt inhibition in cell.

The H+L chain of anti-Her2 monoclonal antibody is at Pichia pastoris bacterial strain GS115 (WT) and genetically engineeredly (express among+Trman) the GS 115 with coexpression T.reesei alpha-Mannosidase.GS115 can (Carlsbad CA) obtains, and except HIS4 suddenlys change to allow the his4 selection, has the phenotype of wild-type basically from Invitrogen.The H+L chain as from two of plasmid pJC284 independently gene express, plasmid pJC284 derives from Invitrogen plasmid pAO815.

The H+L gene uses the anti-Her2 antibody sequence available from GenBank to produce.The GenBank registration number of L chain is 1N8Z A, and the GenBank registration number that the H chain variable region adds the CHI structural domain is 1N8Z B.The GenBank registration number in H chain Fc zone is BC092518.H and L chain DNA sequence are all carried out codon optimized to improve the translation in Pichia pastoris according to Pichia pastoris codon utilization ratio.The optimization of the codon that uses in Pichia sp. is well known in the art, at for example Outchkourove et al., 2002, Protein Expr.Purif.24:18-24; Sharp and Li, 1987, Nucleic Acids Res.15:1281-95; Woo JH, Liuet al., 2002, Protein Expression and Purification 25:270-282 and Nakamura, et al. describes among 2000, the Nucleic Acids Res.28:292.The constant region of H chain (IgG 1) and L chain (human Kappa) is by GeneArt Inc., Regensburg, and Germany is synthetic.(Coralville, oligonucleotide IA) is made with imbrication PCR method oneself available from IDT Inc. in the variable region utilization.Total length H and L chain are synthetic by overlapping PCR, and the H of generation and L chain are cloned into the pCR2.1TOPO carrier (Invitrogen, La Jolla are produced pDX344 and pDX349 respectively in CA).From the H+L chain of pDX344 and pDX349 and GAPDH promotor and AOX1 terminator combined sequence in carrier pDX580 (trunk is from Invitrogen carrier pGAPZA).At last, H+L chain expression cassette from the pDX580 subclone to carrier pJC284.The nucleotides sequence of the codon optimized DNA of coding light chain is listed in shown in the SEQ ID NO:7, and the nucleotides sequence of the codon optimized DNA of encoding heavy chain is listed in shown in the SEQ ID NO:8.Plasmid pJC284 has the GAPDH promotor, be used for to express H+L gene and complete his4 gene, is used for (+Trman) the selection transformant at bacterial strain GS 115 and GS115.Yeast strain GS115 and GS115 (+Trman) transform with pJC285, have the separated bacterial strain of producing anti-Her2 antibody that produces of transformant that is incorporated into the plasmid in the genome at the his4 locus.

(+Trman) structure is as follows for bacterial strain GS115.Trichoderma reesei α-1, the 2-mannosidase expression cassette from plasmid pJC285 is expressed.Plasmid pJC285 is derived from Invitrogen carrier pGAPZA, but it has the Zeocin resistant gene as selective marker, contain the expression cassette that comprises DNA, described dna encoding T.reesei α-1,2-mannosidase catalyst structure domain (SEQ IDNO:9), the be encoded DNA (SEQ ID NO:10) of Saccharomycescerevisiea α MAT front signal sequence of preceding 84 base pairs of its signal sequence of encoding replaces, its ER target amino acid of only encoding, described DNA is at 5 ' end and the DNA that comprises Pichia pastoris GAPH promotor (SEQ ID NO:11), be operably connected with the DNA that comprises Pichia pastoris AOX1 transcription termination sequence (SEQ ID NO:12) at 3 ' end.The nucleotides sequence of expressed intact box is listed among the SEQ ID NO:13 and lists.Yeast strain GS 115 usefulness pJC285 transform, have the GAPDH locus be incorporated into the transformant of the plasmid in the genome separated produce bacterial strain GS 115 (+Trman).

The double culture of bacterial strain is cultivation in the glucose-mixture substratum (BMDY) of 200mL buffering, and described substratum is made up of 1% yeast extract, 2% peptone, 100mM vitriolate of tartar pH6.0,1.34% yeast nitrogen .00004% vitamin H, 2% glucose and the Pmti-2 that is with or without 0.3 or 0.03 μ M.After growth 72 hours, collect culture supernatants, the centrifugal yeast cell of removing.Antibody purifying on the albumin A post in remaining supernatant liquor (about 200mL) fraction, and experience O-glycan analysis as described in Example 2.Except N.F,USP MANNITOL was analyzed, the mean length of the sweet dew sugar chain that O-connects was measured under the situation of hydrolytic action not having by stratographic analysis.The result summarizes in table 2.

There are 14 yeast O-glycan sites at antibody.When producing antibody in wild-type GS 115 bacterial strains, all 14 O-glycan sites all have glycan structures, and only there is a seminose in 8% site, and 39% has two sweet dew sugar chains, and 43% has three sweet dew sugar chains, and 9% has four sweet dew sugar chains (referring to table 2).Yet, when antibody is produced in the wild-type cell of handling with Chemical Inhibition thing Pmti-2,14 O-glycan sites only two occupied, for 76% in these two sites, the sweet dew sugar chain only has a mannose residue.The sweet dew sugar chain that does not all have three or four mannose residues in two sites.Be noted that this analysis can not determine which two of 14 sites are occupied.Any combination in two O-glycan sites of each antibody molecule is occupied, and perhaps specific O-glycan site is preferentially occupied.In the latter's situation, for the antibody that lacks the O-glycan fully (or other protein) is provided, the aminoacid sequence that comprises preferred O-glycan site can be modified to the glycosylated aminoacid sequence of the O-connection of having eliminated described site.

Table 2 has further shown when comprising coding Tricoderma reesei α-1, when producing antibody in the cell of the DNA of 2-mannosidase (bacterial strain GS115 (+Trman)), 14 O-glycan sites only four occupied, for 95% of these four sites, the sweet dew sugar chain only has a mannose residue.Four sites all do not have the sweet dew sugar chain of three or four mannose residues.If specific site is by preferentially O-glycosylation, for the antibody that lacks the O-glycan fully (or other protein) is provided, the aminoacid sequence that comprises preferred O-glycan site can be modified to the glycosylated aminoacid sequence of the O-that has eliminated these site.

At last, table 2 has shown that working as antibody is comprising coding Tricoderma reesei α-1, when producing in the cell of the DNA of 2-mannosidase, under having the situation of Pmti-2,14 O-glycan sites only one occupied, this site for 91%, sweet dew sugar chain only have a mannose residue.There is not the sweet dew sugar chain to have three or four mannose residues.If only there is one or the minority site is only arranged by preferentially O-glycosylation, for the antibody that does not have the O-glycan fully (or other protein) is provided, the aminoacid sequence that comprises described preferred O-glycan site can be modified to and eliminate the glycosylated aminoacid sequence of O-in this site.

Table 2 has shown that further it is a seminose that the Pmti inhibition of 0.3 μ M enough reduces the chain length that takies about 86%, 76% molecule, allows the culture growth simultaneously.Include Tricodermareesei α-1, the 2-mannosidase allows that the quantity of Pmti inhibition reduces by 10 times, and taking the chain length that (occupancy) be reduced to 93%, 87% molecule is a seminose.These results show that using not, the Pmti inhibition of the quantity of cell killing enough produces the glycosylated glycoprotein of the O-connection with reduction.These results show that further the Pmti inhibition appears to reduce synergistically the glycosylated quantity that O-is connected with alpha-Mannosidase.

Table 2

Bacterial strain	Take	Man1	Man2	Man3	Man4
						GS115	14	8	39	43	9
GS115+0.3μM Pmti-2	2	76	24	0	0
						GS115(+Trman)	4	95	5	0	0
GS115(+Trman)+0.3μM Pmti-2	1	91	9	0	0
						GS115(+Trman)+0.03μM Pmti-2	1	87	13	0	0

Above-mentioned each 7 μ L supernatant liquors are reduced and experience SDS-PAGE and Western trace, use anti-IgG (the H ﹠amp of HRP combination; L) detect H and L chain.The result is shown in Figure 2.The glycosylated H chain of super O-is the slowest band of visible migration in the first pair of swimming lane in the accompanying drawing 2.Accompanying drawing 2 has shown as antibody and Tricoderma reesei α-1, during 2-mannosidase coexpression or the cell of expressing antibodies when under the situation that has the Pmti-2 inhibition, hatching or as antibody and Tricoderma reesei α-1, when 2-mannosidase coexpression and the cell of expressing two kinds of protein are hatched, exist the reduction of the glycosylated heavy chain quantity of O-under the situation that has the Pmti-2 inhibition.

Though described the present invention at this with reference to illustrated embodiment, should be understood that to the invention is not restricted to this.Have the ordinary skill of this area and the personnel of acceptance instruction herein and will recognize that other are revised and embodiment is in its scope.Thereby, the claim restriction that the present invention only attaches from here.

Sequence table

<110>GlycoFi，Inc.

Bobrowiza，Piotr

Cook，James W.

Kett，Warren

＜120〉has the generation of the glycosylated glycoprotein of O-of reduction

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<151>2005-11-15

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<170>FastSEQ for Windows Version 4.0

<210>1

<211>288

<212>PRT

＜213〉artificial sequence

<220>

＜223〉Kringle 1-3 albumen

<400>1

Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser

1 5 10 15

Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro

20 25 30

His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu

35 40 45

Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys

50 55 60

Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu

65 70 75 80

Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys

85 90 95

Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln

100 105 110

Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn

115 120 125

Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp

130 135 140

Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro

145 150 155 160

Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu

165 170 175

Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser

180 185 190

Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His Ser

195 200 205

Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys

210 215 220

Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn Ser

225 230 235 240

Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser Pro

245 250 255

Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro

260 265 270

Val Val Gln Asp Gly Gly Gly His His His His His His His His His

275 280 285

<210>2

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer K1-3/UP

<400>2

cggaattctc agagtgcaag actgggaata gaa 33

<210>3

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer K1-3/LP1

<400>3

atgatgatga ccaccaccgt cctggaccac aggggttag 39

<210>4

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer K1-3/LP2

<400>4

ttaatgatga tgatgatgat gatgatgatg accaccacc 39

<210>5

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉forward primer K3f (Asn is to Ser)

<400>5

acccctcaca cacattctag gacaccagaa aacttc 36

<210>6

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reverse primer K3r

<400>6

ctgtgcactc cagtgctgac aggtgtg 27

<210>7

<211>645

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the codon optimized sequence of coding light chain

<400>7

gacattcaga tgacacagtc tccatcttct ttgtccgctt ccgtcggtga tagagttact 60

atcacctgta gagcttccca agacgtcaac accgctgtcg cctggtacca acagaagcca 120

ggtaaggctc caaaactttt gatctactct gcctctttct tgtactccgg tgttccatcc 180

agattttctg gttctagatc cggtaccgac ttcaccttga ccatctcttc cttgcaacca 240

gaagacttcg ctacctacta ctgtcaacaa cactacacta ctcctccaac tttcggtcaa 300

ggaactaagg ttgagattaa gagaactgtt gctgctccat ccgttttcat tttcccacca 360

tccgacgaac aattgaagtc tggtacagct tccgttgttt gtttgttgaa caacttctac 420

ccaagagagg ctaaggttca gtggaaggtt gacaacgctt tgcaatccgg taactcccaa 480

gaatccgtta ctgagcagga ttctaaggat tccacttact ccttgtcctc cactttgact 540

ttgtccaagg ctgattacga gaagcacaag gtttacgcat gcgaggttac acatcagggt 600

ttgtcctccc cagttactaa gtccttcaac agaggagagt gttaa 645

<210>8

<211>1353

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the codon optimized sequence of encoding heavy chain

<400>8

gaggtccaat tggttgaatc tggtggaggt ttggtccaac caggtggatc tctgagactt 60

tcttgtgctg cctctggttt caacattaag gatacttaca tccactgggt tagacaggct 120

ccaggtaagg gtttggagtg ggttgctaga atctacccaa ccaacggtta caccagatac 180

gctgattccg ttaagggtag attcaccatt tccgctgaca cttccaagaa cactgcttac 240

ttgcaaatga actctttgag agctgaggac actgccgtct actactgttc cagatggggt 300

ggtgacggtt tctacgccat ggactactgg ggtcaaggta ccttggttac tgtctcttcc 360

gcttctacta agggaccatc cgtttttcca ttggctccat cctctaagtc tacttccggt 420

ggtactgctg ctttgggatg tttggttaag gactacttcc cagagcctgt tactgtttct 480

tggaactccg gtgctttgac ttctggtgtt cacactttcc cagctgtttt gcaatcttcc 540

ggtttgtact ccttgtcctc cgttgttact gttccatcct cttccttggg tactcagact 600

tacatctgta acgttaacca caagccatcc aacactaagg ttgacaagaa ggttgagcca 660

aagtcctgtg acaagacaca tacttgtcca ccatgtccag ctccagaatt gttgggtggt 720

ccatccgttt tcttgttccc accaaagcca aaggacactt tgatgatctc cagaactcca 780

gaggttacat gtgttgttgt tgacgtttct cacgaggacc cagaggttaa gttcaactgg 840

tacgttgacg gtgttgaagt tcacaacgct aagactaagc caagagagga gcagtacaac 900

tccacttaca gagttgtttc cgttttgact gttttgcacc aggattggtt gaacggaaag 960

gagtacaagt gtaaggtttc caacaaggct ttgccagctc caatcgaaaa gactatctcc 1020

aaggctaagg gtcaaccaag agagccacag gtttacactt tgccaccatc cagagatgag 1080

ttgactaaga accaggtttc cttgacttgt ttggttaaag gattctaccc atccgacatt 1140

gctgttgagt gggaatctaa cggtcaacca gagaacaact acaagactac tccaccagtt 1200

ttggattctg acggttcctt cttcttgtac tccaagttga ctgttgacaa gtccagatgg 1260

caacagggta acgttttctc ctgttccgtt atgcatgagg ctttgcacaa ccactacact 1320

caaaagtcct tgtctttgtc cccaggtaag taa 1353

<210>9

<211>500

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Pichia pastoris GAPDH promotor

<400>9

agatcttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca tctctgaaat 60

atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc cggggtaaaa 120

cttaaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct tccaccgccc 180

gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc cccttgcagc 240

aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg acctagcagc 300

ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg catgtcatga 360

gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca attttggttt 420

ctcctgaccc aaagacttta aatttaattt atttgtccct atttcaatca attgaacaac 480

tatttcgaaa cgagggaatt 500

<210>10

<211>57

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Saccharomyces cerevisiea alpha-MAT front signal sequence

<400>10

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagct 57

<210>11

<211>1494

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Tricoderma reesei alph-1,2-mannosidase catalyst structure domain

<400>11

cgcgccggat ctcccaaccc tacgagggcg gcagcagtca aggccgcatt ccagacgtcg 60

tggaacgctt accaccattt tgcctttccc catgacgacc tccacccggt cagcaacagc 120

tttgatgatg agagaaacgg ctggggctcg tcggcaatcg atggcttgga cacggctatc 180

ctcatggggg atgccgacat tgtgaacacg atccttcagt atgtaccgca gatcaacttc 240

accacgactg cggttgccaa ccaaggcatc tccgtgttcg agaccaacat tcggtacctc 300

ggtggcctgc tttctgccta tgacctgttg cgaggtcctt tcagctcctt ggcgacaaac 360

cagaccctgg taaacagcct tctgaggcag gctcaaacac tggccaacgg cctcaaggtt 420

gcgttcacca ctcccagcgg tgtcccggac cctaccgtct tcttcaaccc tactgtccgg 480

agaagtggtg catctagcaa caacgtcgct gaaattggaa gcctggtgct cgagtggaca 540

cggttgagcg acctgacggg aaacccgcag tatgcccagc ttgcgcagaa gggcgagtcg 600

tatctcctga atccaaaggg aagcccggag gcatggcctg gcctgattgg aacgtttgtc 660

agcacgagca acggtacctt tcaggatagc agcggcagct ggtccggcct catggacagc 720

ttctacgagt acctgatcaa gatgtacctg tacgacccgg ttgcgtttgc acactacaag 780

gatcgctggg tccttgctgc cgactcgacc attgcgcatc tcgcctctca cccgtcgacg 840

cgcaaggact tgaccttttt gtcttcgtac aacggacagt ctacgtcgcc aaactcagga 900

catttggcca gttttgccgg tggcaacttc atcttgggag gcattctcct gaacgagcaa 960

aagtacattg actttggaat caagcttgcc agctcgtact ttgccacgta caaccagacg 1020

gcttctggaa tcggccccga aggcttcgcg tgggtggaca gcgtgacggg cgccggcggc 1080

tcgccgccct cgtcccagtc cgggttctac tcgtcggcag gattctgggt gacggcaccg 1140

tattacatcc tgcggccgga gacgctggag agcttgtact acgcataccg cgtcacgggc 1200

gactccaagt ggcaggacct ggcgtgggaa gcgttcagtg ccattgagga cgcatgccgc 1260

gccggcagcg cgtactcgtc catcaacgac gtgacgcagg ccaacggcgg gggtgcctct 1320

gacgatatgg agagcttctg gtttgccgag gcgctcaagt atgcgtacct gatctttgcg 1380

gaggagtcgg atgtgcaggt gcaggccaac ggcgggaaca aatttgtctt taacacggag 1440

gcgcacccct ttagcatccg ttcatcatca cgacggggcg gccaccttgc ttaa 1494

<210>12

<211>486

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Pichia pastoris AOX1 transcription termination sequence

<400>12

aagggcgaat tcaattcacg tggcccagcc ggccgtctcg gatcggtacc tcgagccgcg 60

gcggccgcca gcttgggccc gaacaaaaac tcatctcaga agaggatctg aatagcgccg 120

tcgaccatca tcatcatcat cattgagttt tagccttaga catgactgtt cctcagttca 180

agttgggcac ttacgagaag accggtcttg ctagattcta atcaagagga tgtcagaatg 240

ccatttgcct gagagatgca ggcttcattt ttgatacttt tttatttgta acctatatag 300

tataggattt tttttgtcat tttgtttctt ctcgtacgag cttgctcctg atcagcctat 360

ctcgcagctg atgaatatct tgtggtaggg gtttgggaaa atcattcgag tttgatgttt 420

ttcttggtat ttcccactcc tcttcagagt acagaagatt aagtgagacc ttcgtttgtg 480

cggatc 486

<210>13

<211>2550

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Tricoderma reesei alpha-1,2-mannosidase expression cassette

＜221〉promotor

<222>(1)...(507)

＜223〉GAPDH promoter sequence

<221>sig_peptide

<222>(508)...(564)

＜223〉coding Saccharomyces cerevisiea alpha-MAT front signal sequence

<221>CDS

<222>(565)...(2058)

＜223〉coding Tricoderma reesei alpha-1,2-mannosidase catalyst structure domain

＜221〉terminator

<222>(2065)...(2550)

＜223〉Pichia pastoris AOX1 transcription termination sequence

<400>13

agatcttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca tctctgaaat 60

atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc cggggtaaaa 120

cttaaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct tccaccgccc 180

gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc cccttgcagc 240

aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg acctagcagc 300

ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg catgtcatga 360

gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca attttggttt 420

ctcctgaccc aaagacttta aatttaattt atttgtccct atttcaatca attgaacaac 480

tatttcgaaa cgagggaatt cgaaacg atg aga ttt cct tca att ttt act gct 534

Met Arg Phe Pro Ser Ile Phe Thr Ala

-15

gtt tta ttc gca gca tcc tcc gca tta gct cgc gcc gga tct ccc aac 582

Val Leu Phe Ala Ala Ser Ser Ala Leu Ala Arg Ala Gly Ser Pro Asn

-10 -5 1 5

cct acg agg gcg gca gca gtc aag gcc gca ttc cag acg tcg tgg aac 630

Pro Thr Arg Ala Ala Ala Val Lys Ala Ala Phe Gln Thr Ser Trp Asn

10 15 20

gct tac cac cat ttt gcc ttt ccc cat gac gac ctc cac ccg gtc agc 678

Ala Tyr His His Phe Ala Phe Pro His Asp Asp Leu His Pro Val Ser

25 30 35

aac agc ttt gat gat gag aga aac ggc tgg ggc tcg tcg gca atc gat 726

Asn Ser Phe Asp Asp Glu Arg Asn Gly Trp Gly Ser Ser Ala Ile Asp

40 45 50

ggc ttg gac acg gct atc ctc atg ggg gat gcc gac att gtg aac acg 774

Gly Leu Asp Thr Ala Ile Leu Met Gly Asp Ala Asp Ile Val Asn Thr

55 60 65 70

atc ctt cag tat gta ccg cag atc aac ttc acc acg act gcg gtt gcc 822

Ile Leu Gln Tyr Val Pro Gln Ile Asn Phe Thr Thr Thr Ala Val Ala

75 80 85

aac caa ggc atc tcc gtg ttc gag acc aac att cgg tac ctc ggt ggc 870

Asn Gln Gly Ile Ser Val Phe Glu Thr Asn Ile Arg Tyr Leu Gly Gly

90 95 100

ctg ctt tct gcc tat gac ctg ttg cga ggt cct ttc agc tcc ttg gcg 918

Leu Leu Ser Ala Tyr Asp Leu Leu Arg Gly Pro Phe Ser Ser Leu Ala

105 110 115

aca aac cag acc ctg gta aac agc ctt ctg agg cag gct caa aca ctg 966

Thr Asn Gln Thr Leu Val Asn Ser Leu Leu Arg Gln Ala Gln Thr Leu

120 125 130

gcc aac ggc ctc aag gtt gcg ttc acc act ccc agc ggt gtc ccg gac 1014

Ala Asn Gly Leu Lys Val Ala Phe Thr Thr Pro Ser Gly Val Pro Asp

135 140 145 150

cct acc gtc ttc ttc aac cct act gtc cgg aga agt ggt gca tct agc 1062

Pro Thr Val Phe Phe Asn Pro Thr Val Arg Arg Ser Gly Ala Ser Ser

155 160 165

aac aac gtc gct gaa att gga agc ctg gtg ctc gag tgg aca cgg ttg 1110

Asn Asn Val Ala Glu Ile Gly Ser Leu Val Leu Glu Trp Thr Arg Leu

170 175 180

agc gac ctg acg gga aac ccg cag tat gcc cag ctt gcg cag aag ggc 1158

Ser Asp Leu Thr Gly Asn Pro Gln Tyr Ala Gln Leu Ala Gln Lys Gly

185 190 195

gag tcg tat ctc ctg aat cca aag gga agc ccg gag gca tgg cct ggc 1206

Glu Ser Tyr Leu Leu Asn Pro Lys Gly Ser Pro Glu Ala Trp Pro Gly

200 205 210

ctg att gga acg ttt gtc agc acg agc aac ggt acc ttt cag gat agc 1254

Leu Ile Gly Thr Phe Val Ser Thr Ser Asn Gly Thr Phe Gln Asp Ser

215 220 225 230

agc ggc agc tgg tcc ggc ctc atg gac agc ttc tac gag tac ctg atc 1302

Ser Gly Ser Trp Ser Gly Leu Met Asp Ser Phe Tyr Glu Tyr Leu Ile

235 240 245

aag atg tac ctg tac gac ccg gtt gcg ttt gca cac tac aag gat cgc 1350

Lys Met Tyr Leu Tyr Asp Pro Val Ala Phe Ala His Tyr Lys Asp Arg

250 255 260

tgg gtc ctt gct gcc gac tcg acc att gcg cat ctc gcc tct cac ccg 1398

Trp Val Leu Ala Ala Asp Ser Thr Ile Ala His Leu Ala Ser His Pro

265 270 275

tcg acg cgc aag gac ttg acc ttt ttg tct tcg tac aac gga cag tct 1446

Ser Thr Arg Lys Asp Leu Thr Phe Leu Ser Ser Tyr Asn Gly Gln Ser

280 285 290

acg tcg cca aac tca gga cat ttg gcc agt ttt gcc ggt ggc aac ttc 1494

Thr Ser Pro Asn Ser Gly His Leu Ala Ser Phe Ala Gly Gly Asn Phe

295 300 305 310

atc ttg gga ggc att ctc ctg aac gag caa aag tac att gac ttt gga 1542

Ile Leu Gly Gly Ile Leu Leu Asn Glu Gln Lys Tyr Ile Asp Phe Gly

315 320 325

atc aag ctt gcc agc tcg tac ttt gcc acg tac aac cag acg gct tct 1590

Ile Lys Leu Ala Ser Ser Tyr Phe Ala Thr Tyr Asn Gln Thr Ala Ser

330 335 340

gga atc ggc ccc gaa ggc ttc gcg tgg gtg gac agc gtg acg ggc gcc 1638

Gly Ile Gly Pro Glu Gly Phe Ala Trp Val Asp Ser Val Thr Gly Ala

345 350 355

ggc ggc tcg ccg ccc tcg tcc cag tcc ggg ttc tac tcg tcg gca gga 1686

Gly Gly Ser Pro Pro Ser Ser Gln Ser Gly Phe Tyr Ser Ser Ala Gly

360 365 370

ttc tgg gtg acg gca ccg tat tac atc ctg cgg ccg gag acg ctg gag 1734

Phe Trp Val Thr Ala Pro Tyr Tyr Ile Leu Arg Pro Glu Thr Leu Glu

375 380 385 390

agc ttg tac tac gca tac cgc gtc acg ggc gac tcc aag tgg cag gac 1782

Ser Leu Tyr Tyr Ala Tyr Arg Val Thr Gly Asp Ser Lys Trp Gln Asp

395 400 405

ctg gcg tgg gaa gcg ttc agt gcc att gag gac gca tgc cgc gcc ggc 1830

Leu Ala Trp Glu Ala Phe Ser Ala Ile Glu Asp Ala Cys Arg Ala Gly

410 415 420

agc gcg tac tcg tcc atc aac gac gtg acg cag gcc aac ggc ggg ggt 1878

Ser Ala Tyr Ser Ser Ile Asn Asp Val Thr Gln Ala Asn Gly Gly Gly

425 430 435

gcc tct gac gat atg gag agc ttc tgg ttt gcc gag gcg ctc aag tat 1926

Ala Ser Asp Asp Met Glu Ser Phe Trp Phe Ala Glu Ala Leu Lys Tyr

440 445 450

gcg tac ctg atc ttt gcg gag gag tcg gat gtg cag gtg cag gcc aac 1974

Ala Tyr Leu Ile Phe Ala Glu Glu Ser Asp Val Gln Val Gln Ala Asn

455 460 465 470

ggc ggg aac aaa ttt gtc ttt aac acg gag gcg cac ccc ttt agc atc 2022

Gly Gly Asn Lys Phe Val Phe Asn Thr Glu Ala His Pro Phe Ser Ile

475 480 485

cgt tca tca tca cga cgg ggc ggc cac ctt gct taa ttaaggaagg 2068

Arg Ser Ser Ser Arg Arg Gly Gly His Leu Ala *

490 495

gcgaattcaa ttcacgtggc ccagccggcc gtctcggatc ggtacctcga gccgcggcgg 2128

ccgccagctt gggcccgaac aaaaactcat ctcagaagag gatctgaata gcgccgtcga 2188

ccatcatcat catcatcatt gagttttagc cttagacatg actgttcctc agttcaagtt 2248

gggcacttac gagaagaccg gtcttgctag attctaatca agaggatgtc agaatgccat 2308

ttgcctgaga gatgcaggct tcatttttga tactttttta tttgtaacct atatagtata 2368

ggattttttt tgtcattttg tttcttctcg tacgagcttg ctcctgatca gcctatctcg 2428

cagctgatga atatcttgtg gtaggggttt gggaaaatca ttcgagtttg atgtttttct 2488

tggtatttcc cactcctctt cagagtacag aagattaagt gagaccttcg tttgtgcgga 2548

tc 2550

Claims (23)

1. A method of producing a protein with reduced O-linked glycosylation comprising: (a) providing a nucleic acid encoding a protein operably linked to an inducible promoter; (b) introducing said nucleic acid into a yeast host cell or a filamentous fungal host cell to provide a culture of said host cell; (c) said culture is grown for a sufficient time to provide said host cells with said nucleic acid in large quantities, thereafter contacting said cultures; and (d) The culture is contacted with an inducer of the promoter to induce expression of the protein for a period of time, followed by or simultaneously with one or more of the Pmt-mediated O-linked glycosylation Inhibitor contacts the culture, (e) isolating a glycoprotein produced by said host cell in the presence of said one or more inhibitors to produce a protein having reduced O-linked glycosylation. 2. The method of claim 1, wherein the one or more inhibitors are benzylidene thiazolidinedione. 3. The method of claim 2, wherein said one or more inhibitors are selected from the group consisting of 5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2 -Thio-3-thiazolidineacetic acid; 5-[[3-(1-phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo -2-thio-3-thiazolidineacetic acid; and 5-[[3-(1-phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]phenylene The group consisting of methyl]-4-oxo-2-thioxo-3-thiazolidineacetic acid. 4. The method of claim 1, wherein the host cell is Pichia pastoris or Saccharomyces cerevisiae . 5. The method of claim 1, wherein said host cell has been genetically modified to produce a glycoprotein having a predominant N-glycan sugar form selected from the group consisting of Man ₅ GlcNAc ₂ , Man ₃ GlcNAc ₂ 、GlcNAcMan ₅ GlcNAc ₂ 、GlcNAcMan ₃ GlcNAc ₂ 、GlcNAc ₂ Man ₃ GlcNAc ₂ 、GalGlcNAcMan ₅ GlcNAc ₂ 、Gal(GlcNAc) ₂ Man ₅ GlcNAc ₂ 、(GalGlcNAc) ₂ Man ₅ GlcNAc ₂ 、NANAGalGlcNAcMan ₃ GlcNAc ₂ , NANA ₂ Gal ₂ GlcNAcMan ₃ GlcNAc ₂ and GalGlcNAcMan ₃ GlcNAc ₂ . 6. The method of claim 1, wherein said host cell has been genetically modified to produce a glycoprotein in which the N-glycosylation pattern is human-like or humanized. 7. The method of claim 1, wherein the glycoprotein is produced at a yield of at least 100 mg/liter of culture medium. 8. A method of producing a protein with reduced O-linked glycosylation comprising: (a) providing a nucleic acid encoding a protein operably linked to an inducible promoter; (b) introducing the nucleic acid into a yeast host cell or a filamentous fungal host cell, and growing a culture of the host cell containing the nucleic acid to produce the host cell for a time sufficient to provide a large amount of the nucleic acid having the nucleic acid said host cell, thereafter contacting said culture with one or more inhibitors of said Pmt-mediated O-linked glycosylation; (c) contacting said culture with one or more inhibitors of Pmt-mediated O-linked glycosylation and optionally one or more α-1,2-mannosidases; and (d) isolating a glycoprotein produced by said host cell in the presence of said one or more inhibitors and said one or more alpha-1,2-mannosidases to produce a protein with reduced O- Linked glycosylated proteins. 9. The method of claim 8, wherein the culture is grown in the presence of one or more inhibitors of Pmt-mediated O-linked glycosylation. 10. The method of claim 8, wherein the culture is grown in the presence of one or more alpha-1,2-mannosidases. 11. The method of claim 8, wherein a second nucleic acid encoding said one or more alpha-1,2-mannosidases is provided and said second nucleic acid is introduced into said host cell. 12. The method of claim 8, wherein a second nucleic acid encoding said one or more α-1,2-mannosidases operably linked to an inducible promoter is provided and said second nucleic acid is introduced into said in the host cells described above. 13. The method of claim 12, wherein said culture is grown for a sufficient time to provide a large number of said host cells before inducing expression of said protein and said one or more alpha-1,2-mannosidases to produce proteins with reduced O-linked glycosylation. 14. The method of claim 12, wherein expression of said protein is induced for a period of time followed by inducing expression of said one or more α-1,2-mannosidases to produce said protein with reduced O-linkages. Glycosylated proteins. 15. The method of claim 8, wherein the one or more inhibitors are benzylidene thiazolidinedione. 16. The method of claim 8, wherein said one or more inhibitors are selected from the group consisting of 5-[[3,4-bis(phenylmethoxy)phenyl]methylene]-4-oxo-2 -Thio-3-thiazolidineacetic acid; 5-[[3-(1-phenylethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo -2-thio-3-thiazolidineacetic acid; and 5-[[3-(1-phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]phenylene The group consisting of methyl]-4-oxo-2-thioxo-3-thiazolidineacetic acid. 17. The method of claim 8, wherein the alpha-1,2-mannosidase is from Trichoderma reesei , Saccharomyces sp. or Aspergillus sp. 18. The method of claim 8, wherein the alpha-1,2-mannosidase is from Trichoderma reesei . 19. The method of claim 8, wherein the host cell comprises a second nucleic acid encoding an alpha-1,2-mannosidase operably linked to an inducible promoter. 20. The method of claim 8, wherein said host cell is selected from the group consisting of K. lactis , Pichia pastoris , Pichia methanolica , and Hansenula . 21. The method of claim 8, wherein said host cell is Pichia pastoris or Saccharomyces cerevisiae . 22. The method of claim 8, wherein said host cell has been genetically modified to produce a glycoprotein having a predominant N-glycan glycoform selected from the group consisting _{of Man5GlcNAc2} , Man ₃ GlcNAc ₂ 、GlcNAcMan ₅ GlcNAc ₂ 、GlcNAcMan ₃ GlcNAc ₂ 、GlcNAc ₂ Man ₃ GlcNAc ₂ 、GalGlcNAcMan ₅ GlcNAc ₂ 、Gal(GlcNAc) ₂ Man ₅ GlcNAc ₂ 、(GalGlcNAc) ₂ Man ₅ GlcNAc ₂ 、NANAGalGlcNAcMan ₃ GlcNAc ₂ , NANA ₂ Gal ₂ GlcNAcMan ₃ GlcNAc ₂ and GalGlcNAcMan ₃ GlcNAc ₂ . 23. The method of claim 8, wherein said host cell has been genetically modified to produce a glycoprotein in which the N-glycosylation pattern is human-like or humanized.

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